Novel peptides and combination of peptides for use in immunotherapy against cll and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/375,070, filed Dec. 9, 2016, which claims the benefit of U.S.Provisional Application Ser. No. 62/265,615, filed 10 Dec. 2015, andGreat Britain Application No. 1521746.6, filed 10 Dec. 2015, the contentof each of these applications is herein incorporated by reference intheir entirety.

This application also is related to PCT/EP2016/078718 filed 24 Nov.2016, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-058006_ST25.txt” createdon 21 Aug. 2018, and 82,279 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm withmorphologically mature, immunologically not completely matured Blymphocytes. CLL is diagnosed when at least 5000 B lymphocytes/μl arepresent in peripheral blood, whereof up to 55% are immature. Abnormal Bcells show a characteristic phenotype expressing CD19, dim CD20, dimCD5, CD23, CD79a and dim IgM or dim IgD (Gribben, 2010).

Two different staging systems are used to classify CLL: The Rai systemand the Binet system. The Rai staging system comprises five stages(0-IV) that classify the disease according to the progressiveaccumulation of abnormal cells. The Binet system uses three stages (A,B, C) to rank the disease according to the number of involved sites(http://www.cancer.gov/cancertopics/pdq/treatment/CLL/Patient).

CLL is primarily a disease of the elderly. The mean age at diagnosis is72 years for sporadic cases and 58 years for familial cases. Theincidence of CLL is higher in males than in females, with a male:femaleratio of about 2:1 (Cartwright et al., 2002).

CLL is the most common leukemia in the Western world where it comprisesabout ⅓rd of all leukemias. Incidence rates are similar in the US andEurope, and estimated new cases are about 16,000 per year. CLL is morecommon in Caucasians than in Africans, rarer in Hispanics and NativeAmericans and seldom in Asians. In people of Asian origin, CLL incidencerates are 3-fold lower than in Caucasians (Gunawardana et al., 2008).

The five-year overall survival for patients with CLL is about 79%(http://www.cancernet/cancer-types/leukernia-chronic-lymphocytic-cll/statistics).The prognosis for individual patients depends on the stage at the timeof diagnosis and the occurrence of several prognostic factors. Followingthe Rai system, patients with stage 0 show a median survival of 10 yearsor more, while patients at stage I/II have a median survival of 7 yearsand patients at stage III/IV 0.75-4 years (Gribben, 2010). Severalfactors are associated with poor prognosis in CLL. These include highexpression levels of ZAP-70 or CD38, an IgVH unmutated status and thecytogenetic aberrations del 17p or del 11q (Gribben, 2010).

While CLL is not curable at present, many patients show only slowprogression of the disease or worsening of symptoms. As patients do notbenefit from an early onset of treatment, the initial approach is “watchand wait” (Richards et al., 1999). For patients with symptomatic orrapidly progressing disease, several treatment options are available.These include chemotherapy, targeted therapy, immune-based therapieslike monoclonal antibodies, CARs and active immunotherapy, and stem celltransplants.

Chemotherapeutic drugs used for CLL treatment are mostly alkylatingagents like chlorambucil and cyclophosphamide or purine analogues likefludarabine. The German CLL Study Group (GCLLSG) CCL4 demonstrated thatfludarabine/cyclophosphamide combinational therapy is superior to solefludarabine treatment (complete remission (CR) of 24% vs. 7%) (Eichhorstet al., 2006).

Ibrutinib and idelalisib are kinase inhibitors that target molecules inthe B-cell receptor signaling cascade. Ibrutinib inhibits Bruton'styrosine kinase (BTK), a src-related cytoplasmic tyrosine kinaseimportant for B-cell maturation, and is used for initial or second-linetherapy (Byrd et al., 2013; O'Brien et al., 2014). Idelalisib is aPI3K-delta inhibitor used in combination with rituximab in refractoryCLL (Furman et al., 2014).

Hematopoietic stem cell transplants (HSCTs) can be considered forpatients with poor prognosis, e.g. patients with del 17p or p53mutations. HSCTs can either be allogeneic, where the transplanted cellsare donated from an HLA-matched person, or autologous, where thepatients' own stem cells are re-infused after chemotherapy (Schetelig etal., 2008).

Monoclonal antibodies are widely used in hematologic malignancies. Thisis due to the knowledge of suitable antigens based on the goodcharacterization of immune cell surface molecules and the accessibilityof tumor cells in blood or bone marrow. Common monoclonal antibodiesused in CLL therapy target either CD20 or CD52. Rituximab, the firstmonoclonal anti-CD20 antibody originally approved by the FDA fortreatment of NHLs, is now widely used in CLL therapy. Combinationaltreatment with rituximab/fludarabine/cyclophosphamide leads to higher CRrates and improved overall survival (OS) than the combinationfludarabine/cyclophosphamide and has become the preferred treatmentoption (GCLLSG CLLR) (Hallek et al., 2008). Ofatumomab targets CD20 andis used for therapy of refractory CLL patients (Wierda et al., 2011).Obinutuzumab is another monoclonal anti-CD20 antibody used in first-linetreatment in combination with chlorambucil (Goede et al., 2014).

Alemtuzumab is an anti-CD52 antibody used for treatment of patients withchemotherapy-resistant disease or patients with poor prognostic factorsas del 17p or p53 mutations (Parikh et al., 2011). Novel monoclonalantibodies target CD37 (otlertuzumab, BI 836826, IMGN529 and(177)Lu-tetulomab) or CD40 (dacetuzumab and lucatumumab) and are testedin pre-clinical settings (Robak and Robak, 2014).

Several completed and ongoing trials are based on engineered autologouschimeric antigen receptor (CAR)-modified T cells with CD19 specificity(Maus et al., 2014). So far, only the minority of patients showeddetectable or persistent CARs. One partial response (PR) and twocomplete responses (CR) have been detected in the CAR T-cell trials byPorter et al. and Kalos et al. (Kalos et al., 2011; Porter et al.,2011).

Active immunotherapy includes the following strategies: gene therapy,whole modified tumor cell vaccines, DC-based vaccines and TAA-derivedpeptide vaccines.

Approaches in gene therapy make use of autologous genetically modifiedtumor cells. These B-CLL cells are transfected withimmuno-(co-)stimulatory genes like IL-2, IL-12, TNF-alpha, GM-CSF, CD80,CD40L, LFA-3 and ICAM-1 to improve antigen presentation and T cellactivation (Carballido et al., 2012). While specific T-cell responsesand reduction in tumor cells are readily observed, immune responses areonly transient.

Several studies have used autologous DCs as antigen presenting cells toelicit anti-tumor responses. DCs have been loaded ex vivo with tumorassociated peptides, whole tumor cell lysate, tumor-derived RNA or DNA.Another strategy uses whole tumor cells for fusion with DCs andgeneration of DC-B-CLL-cell hybrids. Transfected DCs initiated both CD4+and CD8+ T-cell responses (Muller et al., 2004). Fusion hybrids and DCsloaded with tumor cell lysate or apoptotic bodies increasedtumor-specific CD8+ T-cell responses. Patients that showed a clinicalresponse had increased IL-12 serum levels and reduced numbers of Tregs(Palma et al., 2008).

Different approaches use altered tumor cells to initiate or increaseCLL-specific immune responses. An example for this strategy is thegeneration of trioma cells: B-CLL cells are fused to anti-Fc receptorexpressing hybridoma cells that have anti-APC specificity. Trioma cellsinduced CLL-specific T-cell responses in vitro (Kronenberger et al.,2008). Another strategy makes use of irradiated autologous CLL cellswith Bacillus Calmette-Guërin as an adjuvant as a vaccine. Severalpatients showed a reduction in leukocyte levels or stable disease (Huset al., 2008). Besides isolated CLL cells, whole blood from CLL patientshas been used as a vaccine after preparation in a blood treatment unit.The vaccine elicited CLL-specific T-cell responses and led to partialclinical responses or stable disease in several patients (Spaner et al.,2005).

Several TAAs are overexpressed in CLL and are suitable for vaccinations.These include fibromodulin (Mayr et al., 2005), RHAMM/CD168(Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006), hTERT (Counter etal., 1995), the oncofetal antigen-immature laminin receptor protein(OFAiLRP) (Siegel et al., 2003), adipophilin (Schmidt et al., 2004),survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt et al., 2002)and the tumor-derived IgVHCDR3 region (Harig et al., 2001; Carballido etal., 2012).

A phase I clinical trial was conducted using the RHAMM-derived R3peptide as a vaccine. 5 of 6 patients had detectable R3-specific CD8+T-cell responses (Giannopoulos et al., 2010).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and CLL in particular. There is also aneed to identify factors representing biomarkers for cancer in generaland CLL in particular, leading to better diagnosis of cancer, assessmentof prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way, each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 385, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 385,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1 Peptides according to the present invention. J = phospho-serineSEQ ID No. Sequence GeneID(s) Official Gene Symbol(s) 1 AIPPSFASIFL 3507IGHM 2 ALHRPDVYL 3507 IGHM 3 VIAELPPKV 3507 IGHM 4 VIAELPPKVSV 3507 IGHM5 ALIFKIASA 4214 MAP3K1 6 ALDTLEDDMTI 2222 FDFT1 7 ALLERTGYTL 10236,10492 HNRNPR, SYNCRIP 8 ALAASALPALV 6124 RPL4 9 ALCDTLITV 27033 ZBTB3210 FVYGESVEL 27033 ZBTB32 11 ALFTFJPLTV 54665 RSBN1 12 ALGEDEITL 25909,285116 AHCTF1, AHCTF1P1 13 VVDGMPPGV 2969, 2970 GTF2I, GTF2IP1 14ALLRLLPGL 25920 COBRA1 15 ALPEVSVEA 79649 MAP7D3 16 ALPGGAAVAAV 79697C14orf169 17 ALTKTNLQL 9750 FAM65B 18 LLGEFSIKM 9750 FAM65B 19 QVMEKLAAV9750 FAM65B 20 ALVDPGPDFVV 546 ATRX 21 ALWAGLLTL 2208 FCER2 22 ALWDPVIEL27340 UTP20 23 ALYLTEVFL 55024 BANK1 24 AMAGDVVYA 160365 CLECL1 25ATYSGLESQSV 3185 HNRNPF 26 AVLLVLPLV 11322 TMC6 27 AVLGLVWLL 11322 TMC628 AVLHLLLSV 1535 CYBA 29 AVLQAVTAV 58155 PTBP2 30 ELLEGSEIYL 147007,23098 TMEM199, SARM1 31 ELMHGVAGV 165631 PARP15 32 FIDKFTPPV 3117, 3122HLA-DQA1, HLA-DRA 33 FIINSSNIFL 253959 RALGAPA1 34 YLPYIFPNI 253959RALGAPA1 35 FILPSSLYL 81539 SLC38A1 36 FILTHVDQL 257106 ARHGAP30 37FIMEGGAMVL 4174 MCM5 38 FIMEGGAMV 4174 MCM5 39 FLDALLTDV 79915 ATAD5 40FLDEDDMSL 23141 ANKLE2 41 FLDPSLDPLL 58508, 85318 MLL3, BAGE3 42FLEEGGVVTV 5810 RAD1 43 FLLGPEALSFA 55183 RIF1 44 FLLSINDFL 157680VPS13B 45 FLPELPADLEA 11064 CNTRL 46 YIIDSAQAV 11064 CNTRL 47 FLSDQPEPYL160518, 23258 DENND5B, DENND5A 48 FLSPQQPPLL 8621 CDK13 49 FLTDLFAQL55671 SMEK1 50 FLFEPVVKAFL 55671 SMEK1 51 FLVEAPHDWDL 9790 BMS1 52FLVETGFLHV 100268168, 114987, WDR31, MYB 4602 53 FLWQHVELVL 55181 SMG854 FLYPFPLALF 440026 TMEM41B 55 FMEPTLLML 23 ABCF1 56 FVFEAPYTL 139818DOCK11 57 GLSEISLRL 139818 DOCK11 58 YIQQGIFSV 139818 DOCK11 59FVFGDENGTVSL 79084 WDR77 60 FVLDHEDGLNL 4926 NUMA1 61 FVYFIVREV 285527FRYL 62 GIIDGSPRL 80183 KIAA0226L 63 SLAHVAGCEL 80183 KIAA0226L 64KLLESVASA 80183 KIAA0226L 65 GLDDMKANL 5079 PAX5 66 SLAGGLDDMKA 5079PAX5 67 GLDDVTVEV 51019 CCDC53 68 GLDQQFAGLDL 1654 DDX3X 69 GLHQREIFL9922 IQSEC1 70 FVPDTPVGV 9922 IQSEC1 71 GLKHDIARV 388512 CLEC17A 72GLLDAGKMYV 911 CD1C 73 GLLEVISALQL 9816 URB2 74 GLLRASFLL 11344, 54106TWF2, TLR9 75 GLSIFAQDLRL 11344, 54106 TWF2, TLR9 76 GLLRIIPYL 23352UBR4 77 GLLRLTWFL 11214 AKAP13 78 GLPSFLTEV 100532732, 4439 MSH5-SAPCD1,MSH5 79 GLQAKIQEA 23224 SYNE2 80 VLIEDELEEL 23224 SYNE2 81 WLVGQEFEL23224 SYNE2 82 GLQSGVDIGV 1265 CNN2 83 GQGEVLVYV 2316 FLNA 84 GVMDVNTAL391370, 6206 RPS12P4, RPS12 85 HLMLHTAAL 80012 PHC3 86 HLYPGAVTI 55103RALGPS2 87 HQIEAVDGEEL 10567 RABAC1 88 ILDEIGADVQA 29920 PYCR2 89ILDFGTFQL 54832 VPS13C 90 VIADLGLIRV 54832 VPS13C 91 ILDLNTYNV 5336PLCG2 92 ILEPLNPLL 9330 GTF3C3 93 ILFNTQINI 64801 ARV1 94 ILFPLRFTL 9675TTI1 95 ILGYMAHEHKV 6530 SLC6A2 96 ILIDKTSFV 951 CD37 97 LLFATQITL 951CD37 98 SLIKYFLFV 951 CD37 99 ILIFHSVAL 84636 GPR174 100 ILNNEVFAI 26999CYFIP2 101 ILVVIEPLL 23451 SF3B1 102 IQDRAVPSL 3930 LBR 103 KLGGTPAPA5218 CDK14 104 KLILLDTPLFL 253190, 94009 SERHL2, SERHL 105 KLMNDIADI 472ATM 106 FMASHLDYL 472 ATM 107 ILYNLYDLL 472 ATM 108 VIYTLIHYI 472 ATM109 KLWEGLTELV 10235 RASGRP2 110 LLFDHLEPMEL 10235 RASGRP2 111KLWNVAAPLYL 92105 INTS4 112 KTLDVDATYEI 3978 LIG1 113 KVPAEEVLVAV 91782CHMP7 114 LIPEGPPQV 64224 HERPUD2 115 LLFDKLYLL 25885 POLR1A 116LLIGATMQV 23225 NUP210 117 LLILENILL 23225 NUP210 118 RLLILENILL 23225NUP210 119 VLPAEFFEV 23225 NUP210 120 AIDAALTSV 23225 NUP210 121RLYEITIEV 23225 NUP210 122 LLIPVVPGV 55178 RNMTL1 123 LLLAEAELLTL 57724EPG5 124 LLLEETEKQAV 92154, 9788 MTSS1L, MTSS1 125 LLLEIGEVGKLFV 23158TBC1D9 126 LLPEGGITAI 9904 RBM19 127 LLPTAPTTV 55729 ATF7IP 128LLSEEEYHL 9793 CKAP5 129 LLVGTLDVV 23061, 23158 TBC1D9B, TBC1D9 130LLVLIPVYL 23344 ESYT1 131 LQALEVLKI 27304 MOCS3 132 LVYEAIIMV 27250PDCD4 133 YLLSGDISEA 27250 PDCD4 134 MLLEHGITLV 200576 PIKFYVE 135MTAGFSTIAGSV 64078 SLC28A3 136 NLDKLWTLV 389435, 6157 RPL27AP6, RPL27A137 NLIKTVIKL 25816 TNFAIP8 138 NLLDIDAPVTV 5530 PPP3CA 139 NLTDVVEKL120892 LRRK2 140 QIAELPATSV 401145 CCSER1 141 QILSEIVEA 10724 MGEA5 142QLDEPAPQV 387032, 80317 ZKSCAN4, ZKSCAN3 143 QLLDTYFTL 7405 UVRAG 144QLPPFPREL 8498 RANBP3 145 SALDTITTV 3832 KIF11 146 SIIEGPIIKL 23347SMCHD1 147 SILETVATL 253714 MMS22L 148 SIVASLITV 221188 GPR114 149SLDNGGYYI 4067 LYN 150 SLFDQPLSII 117289 TAGAP 151 SLFDSAYGA 2313 FLI1152 SLIRILQTI 160518 DENND5B 153 SLLAELHVL 115352 FCRL3 154 SLLAELHVLTV115352 FCRL3 155 SLMLEVPAL 1756 DMD 156 SLNIGDVQL 973 CD79A 157SLNIRDFTM 147694, 7565 ZNF548, ZNF17 158 SLPEAPLDV 29123 ANKRD11 159SLQEEKLIYV 84668 FAM126A 160 SLSFLVPSL 9631 NUP155 161 SMDDGMINV 7011TEP1 162 SMKDDLENV 3688 ITGB1 163 SQLDISEPYKV 3394 IRF8 164 SVHKGFAFV101060301, 3183, HNRNPC, HNRNPCL1 343069, 440563, 649330 165 TLDDDLDTV51092 SIDT2 166 TLDPNQVSL 85464 SSH2 167 TLDTSKLYV 5923 RASGRF1 168YLLDQSFVM 5923 RASGRF1 169 TLLLGLTEV 55423 SIRPG 170 TLTFRVETV 51379CRLF3 171 TLVPPAALISI 6907 TBL1X 172 TLYDMLASI 10973 ASCC3 173 TVIENIHTI23358 USP24 174 VLAELPIIVV 8295 TRRAP 175 FTVPRVVAV 8295 TRRAP 176VLAEQNIIPSA 54855 FAM46C 177 VLDDRELLL 23178 PASK 178 VLFFNVQEV 57536KIAA1328 179 VLLGLEMTL 1606 DGKA 180 LLKDGPEIGL 1606 DGKA 181 VLLSIPFVSV94103 ORMDL3 182 VLLSVPGPPV 2264 FGFR4 183 VLMPTVYQQGV 54472 TOLLIP 184VLSHNLYTV 50619 DEF6 185 VMDDQRDLI 972 CD74 186 VMDPTKILI 255967 PAN3187 VMDTHLVNI 9779 TBC1D5 188 VMGDIPAAV 4678 NASP 189 VMLEMTPEL 3112,6891 HLA-DOB, TAP2 190 VVMGTVPRL 1729 DIAPH1 191 YIFDGSDGGV 5287 PIK3C2B192 YIQEYLTLL 57674 RNF213 193 YLDLSNNRL 81793 TLR10 194 YLDNVLAEL 1840,23220 DTX1, DTX4 195 YLGGFALSV 6850 SYK 196 YLLLQTYVL 51259 TMEM216 197YLQEVPILTL 1633 DCK 198 YLTFLPAEV 64135 IFIH1 199 YLVELSSLL 80279CDK5RAP3 200 YMFEEVPIVI 8667 EIF3H 201 YQLELHGIEL 5698 PSMB9 202YVDDVFLRV 10347 ABCA7 203 ALLSSQLAL 8925 HERC1 204 GLLQINDKIAL 5000 ORC4205 GLSQANFTL 10288, 10859, 10990, LILRB2, LILRB1, LILRB5, 11024, 11025,LILRA1, LILRB3, LILRA3, 11026, 11027, 23547, LILRA2, LILRA4, LILRP2,79166, 79168 LILRA6 206 HMQDVRVLL 84824 FCRLA 207 IIADLDTTIMFA 7094,83660 TLN1, TLN2 208 ILLKTEGINL 166379 BBS12 209 ILQAELPSL 57508 INTS2210 KLLVQDFFL 8564 KMO 211 LIDVKPLGV 134510 UBLCP1 212 NIIEAINELLV 8892EIF2B2 213 RLLYQLVFL 3566 IL4R 214 RLQELTEKL 255394 TCP11L2 215VMQDIVYKL 84759 PCGF1 216 WLAGDVPAA 24148 PRPF6 217 ALDEPPYLTV 51742ARID4B 218 ALGEEWKGYVV 6194, 728131 RPS6 219 ALLNLLESA 5788 PTPRC 220ALPEILFAKV 643 CXCR5 221 ALVSTIIMV 80228 ORAI2 222 ALWELSLKI 2889RAPGEF1 223 ALWVSQPPEI 259197 NCR3 224 AMEALVVEV 1660 DHX9 225ILQERELLPV 1660 DHX9 226 AMNISVPQV 23387 SIK3 227 FLAEASVMTQL 1445 CSK228 FLGGLSPGV 2968 GTF2H4 229 FLLNLQNCHL 55608 ANKRD10 230 FLQDSKVIFV4772 NFATC1 231 FLYIRQLAI 26155, 401010 NOC2L 232 FMHQIIDQV 79789 CLMN233 GIIDINVRL 51163 DBR1 234 GLDDAEYAL 7376 NR1H2 235 GLDDLLLFL 8914TIMELESS 236 GLLESGRHYL 9744 ACAP1 237 GLQENLDVVV 8881 CDC16 238GLVETELQL 55690 PACS1 239 ILAGEMLSV 7203 CCT3 240 ILARDILEI 5714 PSMD8241 ILGDILLKV 79831 KDM8 242 ILLGIQELL 7329 UBE2I 243 ILPTLEKELFL 79886CAAP1 244 ILQALAVHL 54936 ADPRHL2 245 KIMDYSLLLGV 23396 PIP5K1C 246KLDETGVAL 7155 TOP2B 247 KLKDRLPSI 81606 LBH 248 KTVEPPISQV 57466 SCAF4249 LLPTGVFQV 51131 PHF11 250 LLVQEPDGLMV 404734, 54882 ANKHD1-EIF4EBP3,ANKHD1 251 LLYDNVPGA 11262 SP140 252 NLLDPGSSYLL 5074 PAWR 253NLWSVDGEVTV 92017 SNX29 254 QLIPKLIFL 57705 WDFY4 255 YLFEEAISM 57705WDFY4 256 QLLPVSNVVSV 55374 TMC06 257 RIINGIIISV 84329 HVCN1 258RLDYITAEI 10471 PFDN6 259 RLLDEQFAVL 9026 HIP1R 260 SLDDVEGMSV 80196RNF34 261 SLVEAQGWLV 161176 SYNE3 262 SLWNAGTSV 3495 IGHD 263 SQWEDIHVV55843 ARHGAP15 264 TILDYINVV 89790, 89858 SIGLEC10, SIGLEC12 265TLLADDLEIKL 2124 EVI2B 266 TLLDQLDTQL 8678 BECN1 267 TLLDWQDSL 8289ARID1A 268 TLLQVFHLL 63892 THADA 269 TLTDEQFLV 738 VPS51 270 TVLPVPPLSV5430 POLR2A 271 VIRNIVEAA 100128168, 100996747, RPS26P39, RPS26P32,389472, 441502, RPS26P11, RPS26, 6231, 643003, RPS26P28, RPS26P25,728937, 729188 RPS26P58 272 VLDELPPLI 55832, 91689 CAND1, C22orf32 273VLGEYSYLL 23431 AP4E1 274 VLLEYHIAYL 8087 FXR1 275 VLLFIEHSV 134265,7805 AFAP1L1, LAPTM5 276 VLNDGAPNV 117246 FTSJ3 277 VMILKLPFL 25929GEMIN5 278 YLDDLLPKL 89910 UBE3B 279 YMAPEVVEA 2872 MKNK2 280 YTLDSLYWSV81887 LAS1L

KIAA0226L (also known as C13orf18) encodes KIAA0226-like and is locatedon chromosome 13q14.13 (RefSeq, 2002). KIAA0226L is thought to be atumor suppressor gene and is hyper-methylated in cervical cancer.Re-activation of KIAA0226L leads to decreased cell growth, viability,and colony formation (Huisman et al., 2015; Eijsink et al., 2012;Huisman et al., 2013). The methylation pattern of KIAA0226L can be usedto differ between precursor lesions and normal cervix cancer (Milutin etal., 2015). The methylation pattern of KIAA0226L cannot be used asspecific biomarker for cervical cancer (Sohrabi et al., 2014).Re-activation of KIAA0226L partially de-methylates its promotor regionand also decreases repressive histone methylations (Huisman et al.,2013).

FCRL3, also known as FCRH3 or IRTA3, encodes Fc receptor like 3, one ofseveral FC receptor-like glycoproteins of the immunoglobulin receptorsuperfamily which may play a role in regulation of the immune system(RefSeq, 2002). FCRL3 is up-regulated on CD4+CD26− T cells in Sezarysyndrome patients with a high tumor burden, suggesting that FCRL3expression correlates with a high circulating tumor burden (Wysocka etal., 2014). FCRL3 is up-regulated in chronic lymphocytic leukemia(Poison et al., 2006). FCRL3 is a potential marker with prognosticrelevance in chronic lymphocytic leukemia (Zucchetto et al., 2011)

TABLE 2 Additional peptides according to the present invention with noprior known cancer association. SEQ ID No. Sequence GeneID(s) OfficialGene Symbol(s) 281 NLLDDRGMTAL 146206 RLTPR 282 LLRDGIELV 152926 PPM1K283 ILQPMDIHV 54832 VPS13C 284 LLSAAEPVPA 974 CD79B 285 GVATAGCVNEV 3394IRF8 286 FLLEDLSQKL 2177 FANCD2 287 FLWEEKFNSL 10750 GRAP 288GLAESTGLLAV 50855, 84552 PARD6A, PARD6G 289 ILEEQPMDMLL 84301 DDI2 290LANPHELSL 84301 DDI2 291 ILLNEDDLVTI 57724 EPG5 292 AAALIIHHV 25942SIN3A 293 ALDIMIPMV 4217 MAP3K5 294 ALLDQLHTLL 8202 NCOA3 295 ALLQKLQQL122618 PLD4 296 FIAPTGHSL 23157, 55752 SEPT6, SEPT11 297 FLVEPQEDTRL8237 USP11 298 IILPVEVEV 23505 TMEM131 299 ILEENIPVL 55653 BCAS4 300ILLNPAYDVYL 23225 NUP210 301 AASPIITLV 23225 NUP210 302 ILQDLTFVHL 2889RAPGEF1 303 ILSQPTPSL 29028 ATAD2 304 LAIVPVNTL 23132 RAD54L2 305LLFPQIEGIKI 26263 FBX022 306 VVAEELENV 26263 FBX022 307 LLLTKPTEA 5336PLCG2 308 SLYDVSRMYV 5336 PLCG2 309 ILYGTQFVL 5336 PLCG2 310 LLSTLHLLV64098 PARVG 311 LLVDVEPKV 100129492, 6632      SNRPD1 312 LLYNSTDPTL64005 MY01G 313 LMADLEGLHL 8546 AP3B1 314 LMKDCEAEV 5518 PPP2R1A 315LVYEAPETV 57448 BIRC6 316 MLLEHGITL 200576 PIKFYVE 317 NLLAHIWAL 58513EPS15L1 318 NLQVTQPTV 100505503, 402057, RPS17L, RPS17P16, 442216, 6218RPS17P5, RPS17 319 QVIPQLQTV 6671 SP4 320 TIAPVTVAV 6671 SP4 321RLLEFELAQL 6093 ROCK1 322 SLASIHVPL 23163 GGA3 323 SLDLFNCEV 10541,23520, 646791, ANP32B, ANP32C, 647020, 723972, ANP32BP1, ANP32AP1, 8125ANP32A 324 SLYSALQQA 11190 CEP250 325 TLENGVPCV 23240 KIAA0922 326VLAFLVHEL 11176 BAZ2A 327 VLIKWFPEV 54509 RHOF 328 VLLPQETAEIHL 5292PIM1 329 VLMDGSVKL 11184 MAP4K1 330 VLMWEIYSL 695 BTK 331 VLWELAHLPTL23358 USP24 332 VMIQHVENL 4033 LRMP 333 VTLEFPQLIRV 126382 NR2C2AP 334YLLEEKIASL 51199 NIN 335 YLYQEQYFI 10668 CGRRF1 336 YMAVTTQEV 79840NHEJ1 337 YMYEKESEL 22806 IKZF3 338 FLDMTNWNL 64771 C6orf106 339GLWGTVVNI 25914 RTTN 340 KLLEEICNL 10075 HUWE1 341 LLAELPASVHA 2319FLOT2 342 SLITPLQAV 5591 PRKDC 343 TLLEALDCI 158078, 1915   EEF1A1P5,EEF1A1 344 VLAFENPQV 25852 ARMC8 345 YLIEPDVELQRI 25852 ARMC8 346VLVQVSPSL 23404 EXOSC2 347 YLGPVSPSL 4154 MBNL1 348 ALAKPPVVSV 57634EP400 349 ALATHILSL 11215 AKAP11 350 ALEDRVWEL 285025 CCDC141 351ALSEKLARL 115106 HAUS1 352 ALVFELHYV 57705 WDFY4 353 ATPMPTPSV 55206SBNO1 354 FIMDDPAGNSYL 8882 ZNF259 355 FIWPMLIHI 11325 DDX42 356FLHDHQAEL 259197 NCR3 357 FLIQEIKTL 23352 UBR4 358 FLTDYLNDL 54880 BCOR359 FMQDPMEVFV 10212 DDX39A 360 HLIDTNKIQL 55619 DOCK10 361 ILQEFESKL  2074, 267004 ERCC6, PGBD3 362 ILTELGGFEV 51366 UBR5 363 ITTEVVNELYV57222 ERGIC1 364 KMDWIFHTI 5290 PIK3CA 365 LISPLLLPV 144132 DNHD1 366LLSETCVTI 23469 PHF3 367 NLWSLVAKV 9889 ZBED4 368 QLQPTDALLCV 125950RAVER1 369 RLLDLENSLLGL 54899 PXK 370 SIFASPESV 10285 SMNDC1 371SLADDSVLERL 23195 MDN1 372 SLFGPLPGPGPALV 9595 CYTIP 373 TLLADQGEIRV9219 MTA2 374 VLSVITEEL 55621 TRMT1 375 VLWFKPVEL 55720 TSR1 376VLYNQRVEEI 6730 SRP68 377 VVDGTCVAV 57589 KIAA1432 378 YILGKFFAL 8914TIMELESS 379 YLAELVTPIL 1981 EIF4G1 380 YLDRKLLTL 6850 SYK 381YLLEENKIKL 139818 DOCK11 382 YLLPLLQRL 55794 DDX28 383 YLLREWVNL 23019CNOT1 384 YMIGSEVGNYL 6598 SMARCB1 385 YTIPLAIKL 128869 PIGU

TABLE 3 Peptides useful for e.g. personalized cancer therapies. OfficialSEQ ID No. Sequence GeneID(s) Gene Symbol(s) 386 FLGDYVENL 54832 VPS13C387 YLILSSHQL 1269 CNR2 388 GLLSALENV 1269 CNR2 389 GLAALAVHL 2175 FANCA390 GLEERLYTA 29933 GPR132 391 FLLEREQLL 165055 CCDC138 392 FLWERPTLLV79922 MRM1 393 FVMEGEPPKL 348654 GEN1 394 ILSTEIFGV 79703 C11orf80 395ALYGKLLKL 157680 VPS13B 396 TLLGKQVTL 157680 VPS13B 397 FLPPEHTIVYI 9896FIG4 398 FLAELPGSLSL 5326 PLAGL2 399 ALAAPDIVPAL 79886 CAAP1 400ALFQPHLINV 10097 ACTR2 401 AMADKMDMSL 10189 ALYREF 402 LLVSNLDFGV 10189ALYREF 403 FIMPATVADATAV 23352 UBR4 404 FLQPDLDSL 10514 MYBBP1A 405FLVEKQPPQV 6778 STAT6 406 FLWPKEVEL 146206 RLTPR 407 FMIDASVHPTL 221960,51622  CCZ1B, CCZ1 408 FMVDNEAIYDI 10376, 112714, 113457, TUBA1B,TUBA3E, TUBA3D, 51807, 7277, TUBA8, TUBA4A, TUBA3C, 7278, 7846, 84790TUBA1A, TUBA1C 409 GLDAATATV 9869 SETDB1 410 GLFDGVPTTA 122618 PLD4 411GLFEDVTQPGILL 23140 ZZEF1 412 ILGTEDLIVEV 79719 AAGAB 413 ILLEHGADPNL22852 ANKRD26 414 ILSVVNSQL 80183 KIAA0226L 415 ILVTSIFFL 643 CXCR5 416IMEDIILTL 1656 DDX6 417 IQIGEETVITV 2316 FLNA 418 IVTEIISEI 64151 NCAPG419 LLNEILEQV 64151 NCAPG 420 KLIDDQDISISL 80208 SPG11 421 KLWTGGLDNTV7088, 7090, 7091 TLE1, TLE3, TLE4 422 KQFEGTVEI 675 BRCA2 423 LLIGTDVSL9730 VPRBP 424 LLPPLESLATV 5518 PPP2R1A 425 LLSDVRFVL 53339 BTBD1 426LLWGNLPEI 653820, 729533 FAM72B, FAM72A 427 LLYDAVHIV 2899 GRIK3 428LMYPYIYHV 54954 FAM120C 429 NLLETKLQL 219988 PATL1 430 QLIEKNWLL 56992KIF15 431 RMVAEIQNV 11262 SP140 432 SAVDFIRTL 9263 STK17A 433 SILDRDDIFV8237 USP11 434 SIQQSIERLLV 4809 NHP2L1 435 SLFNQEVQI 100528032, 22914,KLRK1, KLRC4 8302 436 SLFSSLEPQIQPV 23029 RBM34 437 SLEPQIQPV 23029RBM34 438 SLFIGEKAVLL 23029 RBM34 439 SLLDLHTKV 27340 UTP20 440SLLEQGKEPWMV 147949, 163087, ZNF583, ZNF383, ZNF850, 342892, 374899,ZNF829, ZNF527 84503 441 SLLEVNEASSV 149628 PYHIN1 442 SLNDLEKDVMLL 6597SMARCA4 443 SLTIDGIYYV 1659 DHX8 444 SMSGYDQVL 3187, 3188 HNRNPH1,HNRNPH2 445 VIIKGLEEITV 3832 KIF11 446 VILTSSPFL 10800 CYSLTR1 447VLDDSIYLV 57565 KLHL14 448 VMNDRLYAI 57565 KLHL14 449 LLDAMNYHL 57565KLHL14 450 VLGPGPPPL 254359 ZDHHC24 451 VLIEYNFSI 55215 FANCI 452VLLSLLEKV 1130 LYST 453 SLLPLVWKI 1130 LYST 454 VLMDEGAVLTL 54596 L1TD1455 VLPDPEVLEAV 57326 PBXIP1 456 YAAPGGLIGV   1968, 255308 EIF2S3 457YIMEPSIFNTL 51497 TH1L 458 YIQEHLLQI 10625 IVNS1ABP 459 YLDFSNNRL 51284TLR7 460 YLEDGFAYV 5558 PRIM2 461 YLLNLNHLGL 23471 TRAM1 462 YVLPKLYVKL100128168, 100996747, RPS26P39, RPS26P11, 441502, 6231, RPS26, RPS26P28,643003, 644166, RPS26P20, RPS26P15, 644928, 644934, RPS26P50, RPS26P2,646753, 728937, RPS26P25, RPS26P58 729188 463 ALMAVVSGL 55103 RALGPS2464 ALSDETKNNWEV 5591 PRKDC 465 FLYDEIEAEVNL 23191, 26999 CYFIP1, CYFIP2466 FLYDEIEAEV 23191, 26999 CYFIP1, CYFIP2 467 IMQDFPAEIFL 25914 RTTN468 KIQEILTQV 10643 IGF2BP3 469 SLDGTELQL 284114 TMEM102 470 TLTNIIHNL94101 ORMDL1 471 VLLAAGPSAA 23225 NUP210 472 YLFESEGLVL 283431 GAS2L3473 ALADDDFLTV 4173 MCM4 474 ALADLIEKELSV 4850 CNOT4 475 ALDDMISTL 7203CCT3 476 ALITEVVRL 26005 C2CD3 477 ALLDQTKTLAESAL 7094 TLN1 478AMFESSQNVLL 64328 XPO4 479 YLAHFIEGL 64328 XPO4 480 FLAEDPKVTL 60489APOBEC3G 481 FLIDTSASM 203522, 26512  DDX26B, INTS6 482 FLTDLEDLTL 26151NAT9 483 FLTEMVHFI 93517 SDR42E1 484 FLVDGPRVQL 90204 ZSWIM1 485GLLDCPIFL 2177 FANCD2 486 GLLDLPFRVGV 23347 SMCHD1 487 GLSDGNPSL 79684MSANTD2 488 GVYDGEEHSV 4113 MAGEB2 489 HLANIVERL 117854, 445372, TRIM6,TRIM6-TRIM34, 53840 TRIM34 490 ILDEKPVII 23460 ABCA6 491 ILFSEDSTKLFV84916 CIRH1A 492 ILLDDNMQIRL 5261 PHKG2 493 LIIDQADIYL 27042 DIEXF 494LLGDSSFFL 283254 HARBI1 495 LLLDEEGTFSL 27013 CNPPD1 496 LLVEQPPLAGV4773 NFATC2 497 LLWDVPAPSL 1388, 7148 ATF6B, TNXB 498 LTAPPEALLMV 79050NOC4L 499 NLMEMVAQL 55636 CHD7 500 QIITSVVSV 5378 PMS1 501 QLALKVEGV25896 INTS7 502 QLLDETSAITL 10915 TCERG1 503 QLYEEPDTKL 10270 AKAP8 504RLYSGISGLEL 84172 POLR1B 505 RVLEVGALQAV 25885 POLR1A 506 SLLPLDDIVRV3708, 3709 ITPR1, ITPR2 507 SLLTEQDLWTV 90806 ANGEL2 508 SVDSAPAAV 10635RAD51AP1 509 TLQEVVTGV 55750 AGK 510 TVDVATPSV 8924 HERC2 511 VLAYFLPEA4171 MCM2 512 VLISVLQAI 26999 CYFIP2 513 YLIPFTGIVGL 26001 RNF167 514YLLDDGTLVV 8872 CDC123 515 YLMPGFIHL 168400, 55510 DDX53, DDX43 516YLPDIIKDQKA 5496 PPM1G 517 YLQLTQSEL 283237 TTC9C 518 YLTEVFLHVV 55024BANK1 519 YLVEGRFSV 55125 CEP192 520 YLVYILNEL 51202 DDX47 521YLYGQTTTYL 7153 TOP2A 522 YVLTQPPSV 28796, 28815, 28831, IGLV3-21,IGLV2-14, IGLJ3, 3537, 3538 IGLC1, IGLC2

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1V describe relative presentation of various peptides of thepresent disclosure.

FIGS. 2A-2D describe relative expression of various peptides of thepresent disclosure.

FIGS. 3A-3F describe simulation graphs of various peptides of thepresent disclosure.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, acute myelogenousleukemia, bile duct cancer, brain cancer, breast cancer, colorectalcarcinoma, esophageal cancer, gallbladder cancer, gastric cancer,hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkinlymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, renal cell cancer, small cell lung cancer, urinarybladder cancer and uterine cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 385. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 202 (see Table 1), and their uses in theimmunotherapy of CLL, acute myelogenous leukemia, bile duct cancer,brain cancer, breast cancer, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, hepatocellular cancer, Merkel cellcarcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer,ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer,small cell lung cancer, urinary bladder cancer and uterine cancer, andpreferably CLL.

As shown in the following Tables 4A and B, many of the peptidesaccording to the present invention are also found on other tumor typesand can, thus, also be used in the immunotherapy of other indications.Also, refer to FIGS. 1A-1V and Example 1.

TABLE 4A Peptides according to the present invention and their specificuses in other proliferative diseases, especially in other cancerousdiseases. The table shows for selected peptides on which additionaltumor types they were found and either over-presented on more than 5% ofthe measured tumor samples, or presented on more than 5% of the measuredtumor samples with a ratio of geometric means tumor vs normal tissuesbeing larger than 3. Over-presentation is defined as higher presentationon the tumor sample as compared to the normal sample with highestpresentation. Normal tissues against which over-presentation was testedwere: adipose tissue, adrenal gland, blood cells, blood vessel, bonemarrow, brain, breast, esophagus, eye, gallbladder, heart, kidney, largeintestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland,peritoneum, pituitary gland, pleura, salivary gland, skeletal muscle,skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea,ureter, and urinary bladder. SEQ ID No. Sequence Other relevantorgans/diseases 1 AIPPSFASIFL NHL 2 ALHRPDVYL NHL 3 VIAELPPKV NHL 5ALIFKIASA AML, BRCA 7 ALLERTGYTL HCC, NHL 8 ALAASALPALV AML 9 ALCDTLITVNHL 10 FVYGESVEL NHL 12 ALGEDEITL NHL, AML, Melanoma 13 VVDGMPPGV NHL 14ALLRLLPGL NHL, AML 15 ALPEVSVEA NHL, AML, Melanoma 19 QVMEKLAAV NHL 20ALVDPGPDFVV NHL 21 ALWAGLLTL NHL 22 ALWDPVIEL NHL 24 AMAGDVVYA NHL 25ATYSGLESQSV Melanoma 26 AVLLVLPLV NHL 27 AVLGLVWLL NSCLC, NHL, UterineCancer 28 AVLHLLLSV NHL, BRCA 29 AVLQAVTAV SCLC, NHL, AML, Melanoma, OC,Gallbladder Cancer, Bile Duct Cancer 30 ELLEGSEIYL NHL 32 FIDKFTPPV NHL33 FIINSSNIFL NHL 35 FILPSSLYL RCC, NHL, AML, Gallbladder Cancer, BileDuct Cancer 37 FIMEGGAMVL AML 38 FIMEGGAMV NHL 39 FLDALLTDV SCLC, NHL,AML, Melanoma 40 FLDEDDMSL SCLC, NHL, Urinary bladder cancer 42FLEEGGVVTV NHL, Melanoma 43 FLLGPEALSFA NHL, AML 44 FLLSINDFL Melanoma45 FLPELPADLEA NHL, Melanoma 46 YIIDSAQAV NHL 48 FLSPQQPPLL NHL 49FLTDLFAQL NHL 52 FLVETGFLHV Melanoma 55 FMEPTLLML NHL, Urinary bladdercancer 56 FVFEAPYTL NHL, AML 57 GLSEISLRL NHL, AML, Melanoma 58YIQQGIFSV NHL 59 FVFGDENGTVSL NHL, Esophageal Cancer, Urinary bladdercancer, Uterine Cancer 60 FVLDHEDGLNL Melanoma, Esophageal Cancer 62GIIDGSPRL NHL 63 SLAHVAGCEL NHL 64 KLLESVASA NHL 66 SLAGGLDDMKA NHL 67GLDDVTVEV HCC, NHL, Melanoma, Esophageal Cancer, OC, Gallbladder Cancer,Bile Duct Cancer 68 GLDQQFAGLDL RCC, HCC, NHL, Melanoma 69 GLHQREIFL NHL70 FVPDTPVGV NHL 71 GLKHDIARV NHL 72 GLLDAGKMYV NHL, Melanoma 74GLLRASFLL NHL, AML, BRCA 75 GLSIFAQDLRL NHL 76 GLLRIIPYL NHL 78GLPSFLTEV HCC, NHL, AML, Melanoma 79 GLQAKIQEA NHL, Esophageal Cancer,Uterine Cancer 80 VLIEDELEEL NHL 81 WLVGQEFEL NHL 82 GLQSGVDIGV NHL 83GQGEVLVYV SCLC, GC, Esophageal Cancer 86 HLYPGAVTI HCC, NHL 87HQIEAVDGEEL GC, NHL, Esophageal Cancer 88 ILDEIGADVQA PC, NHL 89ILDFGTFQL NHL 91 ILDLNTYNV NHL 92 ILEPLNPLL NHL 93 ILFNTQINI SCLC, NHL,AML, Melanoma, OC 94 ILFPLRFTL NSCLC, RCC, CRC, NHL, Melanoma, OC 96ILIDKTSFV NHL 97 LLFATQITL NHL 98 SLIKYFLFV NHL 99 ILIFHSVAL NHL, AML100 ILNNEVFAI NHL, AML 101 ILVVIEPLL NHL 102 IQDRAVPSL AML 103 KLGGTPAPANHL 105 KLMNDIADI NHL 109 KLWEGLTELV AML 110 LLFDHLEPMEL NHL 111KLWNVAAPLYL OC 112 KTLDVDATYEI HCC, NHL, Melanoma, Esophageal Cancer, OC113 KVPAEEVLVAV NHL 116 LLIGATMQV SCLC, NHL, AML 117 LLILENILL NHL, AML,Melanoma 118 RLLILENILL NHL 119 VLPAEFFEV NHL, AML, Urinary bladdercancer, Uterine Cancer 120 AIDAALTSV NHL, AML 121 RLYEITIEV SCLC, NHL,AML, BRCA, OC 122 LLIPVVPGV NSCLC, NHL 124 LLLEETEKQAV RCC, HCC 125LLLEIGEVGKLFV NHL 126 LLPEGGITAI NHL 127 LLPTAPTTV NHL 130 LLVLIPVYLNHL, Melanoma 133 YLLSGDISEA NHL 136 NLDKLWTLV GC, Esophageal Cancer 137NLIKTVIKL NHL 138 NLLDIDAPVTV SCLC, NHL 139 NLTDVVEKL RCC, NHL 141QILSEIVEA NHL, Melanoma 145 SALDTITTV NHL 146 SIIEGPIIKL NHL, Urinarybladder cancer 147 SILETVATL NHL, AML 148 SIVASLITV NHL, AML 149SLDNGGYYI NHL 150 SLFDQPLSII NHL 151 SLFDSAYGA AML 152 SLIRILQTI HCC 153SLLAELHVL NHL 154 SLLAELHVLTV NHL 155 SLMLEVPAL HCC, NHL 156 SLNIGDVQLNHL 157 SLNIRDFTM AML 158 SLPEAPLDV NHL, AML, Uterine Cancer 159SLQEEKLIYV Brain Cancer, NHL, MCC, Melanoma, Esophageal Cancer, Urinarybladder cancer 160 SLSFLVPSL NHL, AML, Urinary bladder cancer 161SMDDGMINV CRC, NHL, Gallbladder Cancer, Bile Duct Cancer 162 SMKDDLENVNSCLC, SCLC, RCC, HCC, NHL, BRCA, Melanoma, Esophageal Cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer 163 SQLDISEPYKV NHL 164SVHKGFAFV NSCLC, RCC, Brain Cancer, GC, AML, Esophageal Cancer, Urinarybladder cancer, Uterine Cancer 165 TLDDDLDTV NHL 166 TLDPNQVSL AML 167TLDTSKLYV NHL 168 YLLDQSFVM NHL 169 TLLLGLTEV NHL, Melanoma 170TLTFRVETV NHL, Melanoma 172 TLYDMLASI CRC, NHL, AML, Melanoma 175FTVPRVVAV NHL 177 VLDDRELLL NHL 178 VLFFNVQEV SCLC, NHL 179 VLLGLEMTLNHL, Esophageal Cancer 180 LLKDGPEIGL NHL 181 VLLSIPFVSV HCC, NHL 183VLMPTVYQQGV AML 185 VMDDQRDLI AML 186 VMDPTKILI AML, Urinary bladdercancer 187 VMDTHLVNI NHL, Urinary bladder cancer 188 VMGDIPAAV NHL, AML,Gallbladder Cancer, Bile Duct Cancer 189 VMLEMTPEL SCLC, PC, NHL, BRCA,Melanoma, Gallbladder Cancer, Bile Duct Cancer 190 VVMGTVPRL NHL, AML192 YIQEYLTLL NHL 193 YLDLSNNRL NHL 194 YLDNVLAEL NHL 195 YLGGFALSV NHL,AML 196 YLLLQTYVL NHL 197 YLQEVPILTL NHL, Melanoma 199 YLVELSSLL HCC,NHL, AML 200 YMFEEVPIVI NSCLC, SCLC, CRC, HCC, NHL, Melanoma, EsophagealCancer, OC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer201 YQLELHGIEL Esophageal Cancer 202 YVDDVFLRV Urinary bladder cancer203 ALLSSQLAL NHL 204 GLLQINDKIAL SCLC, RCC, HCC, Melanoma, OC, Urinarybladder cancer 205 GLSQANFTL NHL 207 IIADLDTTIMFA SCLC, NHL, Melanoma,OC 208 ILLKTEGINL NHL, BRCA, OC, Urinary bladder cancer 209 ILQAELPSLNHL 210 KLLVQDFFL HCC, NHL 211 LIDVKPLGV NHL, BRCA, Melanoma 212NIIEAINELLV NHL, BRCA, Melanoma 213 RLLYQLVFL NHL, Esophageal Cancer,Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer 214RLQELTEKL NHL, AML 215 VMQDIVYKL NHL, Esophageal Cancer, Urinary bladdercancer 216 WLAGDVPAA SCLC, NHL 217 ALDEPPYLTV NHL, Melanoma 218ALGEEWKGYVV Esophageal Cancer 219 ALLNLLESA NHL, AML, OC 220 ALPEILFAKVNHL 221 ALVSTIIMV NHL, AML 222 ALWELSLKI NHL 223 ALWVSQPPEI NHL 224AMEALVVEV NHL 225 ILQERELLPV NHL, Melanoma, OC, Urinary bladder cancer227 FLAEASVMTQL NHL 228 FLGGLSPGV NHL, Esophageal Cancer 229 FLLNLQNCHLNHL 230 FLQDSKVIFV NHL 231 FLYIRQLAI AML 232 FMHQIIDQV RCC, CRC,Melanoma 233 GIIDINVRL PC, NHL, Melanoma, Esophageal Cancer, Urinarybladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 234GLDDAEYAL NHL, AML, Melanoma, Urinary bladder cancer 235 GLDDLLLFL SCLC,PC, NHL, AML, Urinary bladder cancer, Uterine Cancer, GallbladderCancer, Bile Duct Cancer 236 GLLESGRHYL NHL, AML 237 GLQENLDVVV SCLC,NHL, Melanoma, Uterine Cancer 238 GLVETELQL NHL, AML 239 ILAGEMLSV NHL,AML, Melanoma, Gallbladder Cancer, Bile Duct Cancer 240 ILARDILEI NHL,Urinary bladder cancer 241 ILGDILLKV AML 242 ILLGIQELL NHL, AML 243ILPTLEKELFL AML 244 ILQALAVHL NHL, AML, OC 245 KIMDYSLLLGV SCLC, RCC, OC246 KLDETGVAL HCC, NHL, AML, Melanoma 247 KLKDRLPSI NHL 248 KTVEPPISQVNHL 249 LLPTGVFQV Urinary bladder cancer 250 LLVQEPDGLMV SCLC, HCC,Melanoma, Urinary bladder cancer 251 LLYDNVPGA NHL 252 NLLDPGSSYLLEsophageal Cancer 253 NLWSVDGEVTV NHL 254 QLIPKLIFL NHL, Melanoma 255YLFEEAISM NHL 257 RIINGIIISV NHL 258 RLDYITAEI RCC, NHL, AML, Melanoma260 SLDDVEGMSV NHL, Gallbladder Cancer, Bile Duct Cancer 261 SLVEAQGWLVNHL 263 SQWEDIHVV NHL, AML 264 TILDYINVV Brain Cancer, NHL, AML, BRCA,OC, Uterine Cancer 265 TLLADDLEIKL Esophageal Cancer 266 TLLDQLDTQL NHL,OC, Urinary bladder cancer 267 TLLDWQDSL NHL, AML, Melanoma 268TLLQVFHLL NHL 269 TLTDEQFLV NHL, AML 271 VIRNIVEAA GC, CRC, PC, AML,Esophageal Cancer 272 VLDELPPLI SCLC, NHL 273 VLGEYSYLL NHL 274VLLEYHIAYL NSCLC, SCLC, HCC, NHL, Urinary bladder cancer 275 VLLFIEHSVNHL 276 VLNDGAPNV NSCLC, SCLC, NHL, Melanoma, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer 278 YLDDLLPKL PC, NHL 280YTLDSLYWSV NHL 281 NLLDDRGMTAL NHL, AML 282 LLRDGIELV NHL, Melanoma, OC283 ILQPMDIHV NHL 284 LLSAAEPVPA NHL 286 FLLEDLSQKL NHL 288 GLAESTGLLAVNHL 290 LANPHELSL AML 291 ILLNEDDLVTI SCLC, NHL, Melanoma 292 AAALIIHHVUrinary bladder cancer 294 ALLDQLHTLL NHL 295 ALLQKLQQL NHL 296FIAPTGHSL SCLC, NHL, AML, BRCA 297 FLVEPQEDTRL NHL, Melanoma 298IILPVEVEV SCLC, NHL, AML, Urinary bladder cancer 299 ILEENIPVL NHL 300ILLNPAYDVYL NHL, Melanoma 303 ILSQPTPSL NHL, AML, Melanoma, OC 304LAIVPVNTL AML 305 LLFPQIEGIKI NHL, Melanoma 306 VVAEELENV NHL, Melanoma,OC 307 LLLTKPTEA NHL 308 SLYDVSRMYV NHL 309 ILYGTQFVL NHL 310 LLSTLHLLVNHL 311 LLVDVEPKV NSCLC, SCLC, RCC, HCC, PC, NHL, AML, Melanoma 312LLYNSTDPTL NHL 313 LMADLEGLHL Melanoma, Urinary bladder cancer 314LMKDCEAEV NHL 316 MLLEHGITL NHL 317 NLLAHIWAL AML 320 TIAPVTVAV NHL 321RLLEFELAQL SCLC, CRC, NHL, OC 322 SLASIHVPL NHL 323 SLDLFNCEV NHL 324SLYSALQQA NHL 325 TLENGVPCV NHL 326 VLAFLVHEL Esophageal Cancer 327VLIKWFPEV NHL 328 VLLPQETAEIHL NHL, BRCA, Melanoma, OC, Urinary bladdercancer 329 VLMDGSVKL NHL 330 VLMWEIYSL NHL 332 VMIQHVENL NHL 333VTLEFPQLIRV SCLC, HCC, NHL 334 YLLEEKIASL NHL 335 YLYQEQYFI SCLC, NHL,AML, Urinary bladder cancer 337 YMYEKESEL NHL 338 FLDMTNWNL HCC, NHL,Melanoma, OC, Urinary bladder cancer 339 GLWGTVVNI NHL, AML, Melanoma,Uterine Cancer 341 LLAELPASVHA HCC, NHL, Urinary bladder cancer 342SLITPLQAV NHL, AML, Urinary bladder cancer, Gallbladder Cancer, BileDuct Cancer 343 TLLEALDCI NHL, AML, Melanoma, Esophageal Cancer 344VLAFENPQV Esophageal Cancer 346 VLVQVSPSL RCC, NHL, AML, GallbladderCancer, Bile Duct Cancer 347 YLGPVSPSL NHL 348 ALAKPPVVSV SCLC, HCC,Melanoma, Esophageal Cancer 349 ALATHILSL NHL, Urinary bladder cancer350 ALEDRVWEL NHL, Esophageal Cancer 351 ALSEKLARL HCC, NHL, AML,Urinary bladder cancer 352 ALVFELHYV NHL 353 ATPMPTPSV SCLC, NHL 354FIMDDPAGNSYL CRC, NHL, AML, Melanoma, Esophageal Cancer, Urinary bladdercancer, Gallbladder Cancer, Bile Duct Cancer 357 FLIQEIKTL NHL, OC,Urinary bladder cancer 358 FLTDYLNDL NHL 359 FMQDPMEVFV Melanoma 360HLIDTNKIQL NHL 362 ILTELGGFEV NHL 363 ITTEVVNELYV NHL, Melanoma 364KMDWIFHTI Urinary bladder cancer 365 LISPLLLPV NHL, AML, Melanoma,Urinary bladder cancer 367 NLWSLVAKV NSCLC, NHL, AML, Melanoma, OC 369RLLDLENSLLGL AML 370 SIFASPESV Melanoma, Esophageal Cancer, Urinarybladder cancer 371 SLADDSVLERL RCC, HCC, NHL, AML, Esophageal Cancer,Urinary bladder cancer 373 TLLADQGEIRV NHL, Melanoma 374 VLSVITEEL NHL,AML 375 VLWFKPVEL NSCLC, HCC, NHL, Melanoma, Esophageal Cancer 376VLYNQRVEEI Urinary bladder cancer 377 VVDGTCVAV NHL, Melanoma 378YILGKFFAL NHL, AML 379 YLAELVTPIL NSCLC, SCLC, NHL, Melanoma 380YLDRKLLTL NHL 381 YLLEENKIKL NHL 382 YLLPLLQRL NHL, AML 383 YLLREWVNLHCC, NHL 384 YMIGSEVGNYL NHL, Urinary bladder cancer 385 YTIPLAIKL NHL,AML, BRCA, Esophageal Cancer NSCLC = non-small cell lung cancer, SCLC= small cell lung cancer, RCC = kidney cancer, CRC = colon or rectumcancer, GC = stomach cancer, HCC = liver cancer, PC = pancreatic cancer,PrC = prostate cancer, leukemia, BRCA = breast cancer, MCC = Merkel cellcarcinoma, NHL = Non Hodgkin lymphoma

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 2, 3, 7, 9, 10, 13, 14, 15, 19, 20, 21, 22, 24, 26,27, 28, 29, 30, 32, 33, 35, 38, 39, 40, 42, 43, 45, 46, 48, 49, 55, 56,57, 58, 59, 62, 63, 64, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 78, 79,80, 81, 82, 86, 87, 88, 89, 91, 92, 93, 94, 96, 97, 98, 99, 100, 101,103, 105, 110, 112, 113, 116, 117, 118, 119, 120, 121, 122, 125, 126,127, 130, 133, 137, 138, 139, 141, 145, 146, 147, 148, 149, 150, 153,154, 155, 156, 158, 159, 160, 161, 162, 163, 165, 167, 168, 169, 170,172, 175, 177, 178, 179, 180, 181, 187, 188, 189, 190, 192, 193, 194,195, 196, 197, 199, 200, 203, 205, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 219, 220, 221, 222, 223, 224, 225, 227, 228, 229,230, 233, 234, 235, 236, 237, 238, 239, 240, 242, 244, 246, 247, 248,251, 253, 254, 255, 257, 258, 260, 261, 263, 264, 266, 267, 268, 269,272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 286, 288, 290,291, 294, 295, 296, 297, 298, 299, 300, 303, 305, 306, 307, 308, 309,310, 311, 312, 314, 316, 320, 321, 322, 323, 324, 325, 327, 328, 329,330, 332, 333, 334, 335, 337, 338, 339, 341, 342, 343, 346, 347, 349,350, 351, 352, 353, 354, 357, 358, 360, 362, 363, 365, 367, 371, 373,374, 375, 377, 378, 379, 380, 381, 382, 383, 384, and 385 for the—in onepreferred embodiment combined—treatment of NHL.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 5, 8, 12, 14, 15, 29, 35, 37, 39, 43, 56, 57, 74, 78,93, 99, 100, 102, 109, 116, 117, 119, 120, 121, 147, 148, 151, 157, 158,160, 164, 166, 172, 183, 185, 186, 188, 190, 195, 199, 214, 219, 221,231, 234, 235, 236, 238, 239, 241, 242, 243, 244, 246, 258, 263, 264,267, 269, 271, 281, 290, 296, 298, 303, 304, 311, 317, 335, 339, 342,343, 346, 351, 354, 365, 367, 369, 374, 378, 382, and 385 for the—in onepreferred embodiment combined—treatment of AML.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 5, 28, 74, 121, 162, 208, 211, 212, 264, 296, 328, and385 for the—in one preferred embodiment combined—treatment of BRCA.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 67, 68, 78, 86, 112, 124, 152, 155, 162, 181, 199,200, 204, 210, 246, 250, 274, 311, 333, 338, 341, 348, 351, 371, 375,and 383 for the—in one preferred embodiment combined—treatment of HCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 12, 15, 25, 29, 39, 42, 44, 45, 52, 57, 60, 67, 68,72, 78, 93, 94, 112, 117, 130, 141, 159, 162, 169, 170, 172, 189, 197,200, 204, 207, 211, 212, 217, 225, 232, 233, 234, 237, 239, 246, 250,254, 258, 267, 276, 282, 291, 297, 300, 303, 305, 306, 311, 313, 328,338, 339, 343, 348, 354, 359, 363, 365, 367, 370, 373, 375, 377, and 379for the—in one preferred embodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 27, 94, 122, 162, 164, 200, 274, 276, 311, 367, 375,and 379 for the—in one preferred embodiment combined—treatment of NSCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 27, 59, 79, 119, 158, 162, 164, 233, 235, 237, 264,276, and 339 for the—in one preferred embodiment combined—treatment ofuterine cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 29, 67, 93, 94, 111, 112, 200, 204, 207, 208, 219,225, 244, 245, 264, 266, 282, 303, 306, 321, 328, 338, 357, and 367 forthe—in one preferred embodiment combined—treatment of ovarian cancer(OC).

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 29, 35, 67, 161, 162, 188, 189, 200, 213, 233, 235,239, 260, 276, 342, 346, and 354 for the—in one preferred embodimentcombined—treatment of gallbladder cancer and/or bile duct cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 35, 68, 94, 124, 139, 162, 164, 197, 204, 232, 245,258, 311, 346, and 371 for the—in one preferred embodimentcombined—treatment of RCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 39, 40, 83, 93, 121, 138, 162, 178, 189, 200, 204,207, 216, 235, 237, 245, 250, 272, 274, 276, 291, 296, 298, 311, 321,333, 335, 348, 353, and 379 for the—in one preferred embodimentcombined—treatment of SCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 40, 55, 59, 119, 146, 159, 160, 164, 186, 187, 200,202, 204, 208, 213, 215, 225, 233, 234, 235, 240, 249, 250, 266, 274,292, 298, 313, 328, 335, 338, 341, 342, 349, 351, 354, 357, 364, 365,370, 371, 376, and 384 for the—in one preferred embodimentcombined—treatment of urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 59, 60, 67, 79, 83, 87, 112, 136, 159, 162, 164, 179,200, 201, 213, 215, 218, 228, 233, 252, 265, 271, 326, 343, 344, 348,350, 354, 370, 371, 375, and 385 for the—in one preferred embodimentcombined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 83, 87, 136, 164, and 271 for the—in one preferredembodiment combined—treatment of gastric cancer (GC).

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 88, 189, 233, 235, 271, 278, and 311 for the—in onepreferred embodiment combined—treatment of pancreatic cancer (PC).

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 94, 161, 172, 200, 232, 271, 321, and 354 for the—inone preferred embodiment combined—treatment of colorectal cancer (CRC).

Thus, another aspect of the present invention relates to the use of thepeptide according to the present invention according to SEQ ID No. 159for the treatment of MCC.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofCLL, acute myelogenous leukemia, bile duct cancer, brain cancer, breastcancer, colorectal carcinoma, esophageal cancer, gallbladder cancer,gastric cancer, hepatocellular cancer, Merkel cell carcinoma, melanoma,non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, renal cell cancer, small cell lungcancer, urinary bladder cancer and uterine cancer.

TABLE 4B Peptides according to the present invention and their specificuses in other proliferative diseases, especially in other cancerousdiseases. The table shows, like Table 4A, for selected peptides on whichadditional tumour types they were found showing over-presentation(including specific presentation) on more than 5% of the measured tumoursamples, or presentation on more than 5% of the measured tumour sampleswith a ratio of geometric means tumour vs normal tissues being largerthan 3. Over-presentation is defined as higher presentation on thetumour sample as compared to the normal sample with highestpresentation. Normal tissues against which over-presentation was testedwere: adipose tissue, adrenal gland, artery, bone marrow, brain, centralnerve, colon, duodenum, oesophagus, eye, gallbladder, heart, kidney,liver, lung, lymph node, mononuclear white blood cells, pancreas,parathyroid gland, peripheral nerve, peritoneum, pituitary, pleura,rectum, salivary gland, skeletal muscle, skin, small intestine, spleen,stomach, thyroid gland, trachea, ureter, urinary bladder, vein. SEQ IDNO. Sequence Additional Entities 5 ALIFKIASA Uterine Cancer 7 ALLERTGYTLUterine Cancer, AML 8 ALAASALPALV AML, HNSCC 11 ALFTFSPLTV AML, HNSCC,NHL, NSCLC, OC, SCLC 13 VVDGMPPGV BRCA, Uterine Cancer 14 ALLRLLPGLBRCA, Melanoma, HNSCC 15 ALPEVSVEA Esophageal Cancer 25 ATYSGLESQSV AML26 AVLLVLPLV NHL, NSCLC 27 AVLGLVWLL HNSCC 29 AVLQAVTAV Uterine Cancer30 ELLEGSEIYL CRC, BRCA, Melanoma, Uterine Cancer 35 FILPSSLYL SCLC,Uterine Cancer 37 FIMEGGAMVL NHL 38 FIMEGGAMV HNSCC 40 FLDEDDMSL HNSCC41 FLDPSLDPLL Uterine Cancer 42 FLEEGGVVTV SCLC, AML 44 FLLSINDFL RCC,Uterine Cancer, AML, NHL 45 FLPELPADLEA NSCLC, Uterine Cancer, OC 48FLSPQQPPLL HNSCC 49 FLTDLFAQL NHL, Melanoma 51 FLVEAPHDWDL UterineCancer 55 FMEPTLLML HNSCC 58 YIQQGIFSV NHL, NSCLC 59 FVFGDENGTVSL SCLC,HNSCC 67 GLDDVTVEV AML 68 GLDQQFAGLDL BRCA, OC, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, HNSCC 76 GLLRIIPYL RCC 82GLQSGVDIGV AML, Esophageal Cancer, PC 83 GQGEVLVYV NHL 84 GVMDVNTAL AML,HCC, HNSCC, NHL, NSCLC, GC, RCC 86 HLYPGAVTI AML 87 HQIEAVDGEEL PC 89ILDFGTFQL BRCA 90 VIADLGLIRV Melanoma 91 ILDLNTYNV AML 93 ILFNTQINIUterine Cancer, HNSCC 94 ILFPLRFTL Gallbladder Cancer, Bile Duct Cancer,AML, HNSCC 101 ILVVIEPLL Uterine Cancer 107 ILYNLYDLL Melanoma 108VIYTLIHYI BRCA, Melanoma 111 KLWNVAAPLYL Melanoma 112 KTLDVDATYEIUterine Cancer 113 KVPAEEVLVAV NHL, NSCLC 114 LIPEGPPQV AML, HNSCC 115LLFDKLYLL Uterine Cancer 118 RLLILENILL HNSCC 119 VLPAEFFEV SCLC 124LLLEETEKQAV HNSCC 125 LLLEIGEVGKLFV BRCA 126 LLPEGGITAI SCLC, HCC,Melanoma, Uterine Cancer, HNSCC 127 LLPTAPTTV SCLC 128 LLSEEEYHL UrinaryBladder Cancer, NHL, HNSCC 130 LLVLIPVYL Uterine Cancer 136 NLDKLWTLVAML, CRC, Melanoma, NSCLC, Brain Cancer 138 NLLDIDAPVTV Uterine Cancer,AML 141 QILSEIVEA Uterine Cancer 145 SALDTITTV NSCLC, RCC, GC, HCC, AML146 SIIEGPIIKL HNSCC 147 SILETVATL Melanoma 152 SLIRILQTI SCLC 156SLNIGDVQL AML 159 SLQEEKLIYV HNSCC 160 SLSFLVPSL Uterine Cancer 161SMDDGMINV SCLC, Melanoma, Uterine Cancer, AML, HNSCC 162 SMKDDLENV PC,OC, HNSCC 164 SVHKGFAFV CRC, Melanoma, HNSCC 172 TLYDMLASI SCLC, UrinaryBladder Cancer, Uterine Cancer 173 TVIENIHTI CRC, HCC, Melanoma, NHL,NSCLC, OC, Esophageal Cancer, GC, RCC 174 VLAELPIIVV NSCLC, OC 176VLAEQNIIPSA NSCLC, SCLC, GC, Melanoma, NHL, HNSCC 179 VLLGLEMTL HNSCC181 VLLSIPFVSV Melanoma, Gallbladder Cancer, Bile Duct Cancer 184VLSHNLYTV NHL 187 VMDTHLVNI CRC, Melanoma, Uterine Cancer 188 VMGDIPAAVUrinary Bladder Cancer, HNSCC 189 VMLEMTPEL HNSCC 190 VVMGTVPRL Melanoma191 YIFDGSDGGV Uterine Cancer 192 YIQEYLTLL SCLC, Urinary BladderCancer, AML, HNSCC 197 YLQEVPILTL Uterine Cancer 198 YLTFLPAEV BRCA,Uterine Cancer 199 YLVELSSLL Melanoma 200 YMFEEVPIVI BRCA, HNSCC 201YQLELHGIEL NHL 202 YVDDVFLRV AML, HNSCC 203 ALLSSQLAL NHL, AML, NSCLC204 GLLQINDKIAL BRCA, Uterine Cancer, AML 208 ILLKTEGINL Melanoma, HNSCC211 LIDVKPLGV RCC, Brain Cancer, HCC, Esophageal Cancer, HNSCC 212NIIEAINELLV Uterine Cancer, AML, HNSCC 215 VMQDIVYKL HNSCC 216 WLAGDVPAAAML, HNSCC 217 ALDEPPYLTV AML 218 ALGEEWKGYVV AML 221 ALVSTIIMV BrainCancer 224 AMEALVVEV AML 226 AMNISVPQV AML, PrC, Brain Cancer,Gallbladder Cancer, CCC, HNSCC, NHL, NSCLC, OC, SCLC, Uterine Cancer 228FLGGLSPGV SCLC, BRCA, Uterine Cancer, AML, HNSCC 231 FLYIRQLAI UterineCancer 232 FMHQIIDQV Uterine Cancer 234 GLDDAEYAL Uterine Cancer, HNSCC235 GLDDLLLFL BRCA, HNSCC 237 GLQENLDVVV AML 239 ILAGEMLSV SCLC, HCC 240ILARDILEI Melanoma 242 ILLGIQELL BRCA 247 KLKDRLPSI HCC 257 RIINGIIISVAML 258 RLDYITAEI GC, Uterine Cancer 260 SLDDVEGMSV SCLC, HNSCC 261SLVEAQGWLV AML 264 TILDYINVV HNSCC 266 TLLDQLDTQL CRC, BRCA, UterineCancer 268 TLLQVFHLL Uterine Cancer 269 TLTDEQFLV BRCA 272 VLDELPPLIBRCA, Uterine Cancer, HNSCC 273 VLGEYSYLL NHL, HNSCC 274 VLLEYHIAYLMelanoma, HNSCC 275 VLLFIEHSV Melanoma 279 YMAPEVVEA HNSCC, NHL, NSCLC,SCLC, Uterine Cancer 280 YTLDSLYWSV SCLC, BRCA, Melanoma, HNSCC 286FLLEDLSQKL Uterine Cancer 289 ILEEQPMDMLL Uterine Cancer 293 ALDIMIPMVAML, CRC, Brain Cancer, Gallbladder Cancer, CCC, HNSCC, Melanoma, NHL,NSCLC, OC, Brain Cancer, RCC, SCLC, Urinary Bladder Cancer, UterineCancer 294 ALLDQLHTLL AML 298 IILPVEVEV CRC, HNSCC 299 ILEENIPVL UterineCancer, AML 303 ILSQPTPSL Uterine Cancer 305 LLFPQIEGIKI Urinary BladderCancer, HNSCC 306 VVAEELENV RCC 311 LLVDVEPKV Uterine Cancer, HNSCC 312LLYNSTDPTL Urinary Bladder Cancer 313 LMADLEGLHL Uterine Cancer, AML 317NLLAHIWAL NHL 321 RLLEFELAQL Brain Cancer, PrC, Uterine Cancer, AML,HNSCC 323 SLDLFNCEV Uterine Cancer, AML 324 SLYSALQQA SCLC, UterineCancer, AML 326 VLAFLVHEL Esophageal Cancer, NSCLC, RCC 327 VLIKWFPEVUterine Cancer 333 VTLEFPQLIRV BRCA 334 YLLEEKIASL AML 335 YLYQEQYFICRC, Brain Cancer, Gallbladder Cancer, CCC, NSCLC, UterineCancer 336YMAVTTQEV Uterine Cancer 339 GLWGTVVNI HNSCC 340 KLLEEICNL Melanoma,Uterine Cancer, AML, NHL 342 SLITPLQAV HNSCC 343 TLLEALDCI GC, UterineCancer, HNSCC 344 VLAFENPQV HNSCC 346 VLVQVSPSL HNSCC 348 ALAKPPVVSVNSCLC, Urinary Bladder Cancer, NHL, HNSCC 349 ALATHILSL HCC, Melanoma351 ALSEKLARL CRC, HNSCC, Melanoma, NSCLC, OC, PC, Brain Cancer, RCC,Uterine Cancer 353 ATPMPTPSV BRCA, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer, AML, HNSCC 354 FIMDDPAGNSYL Brain Cancer, HNSCC 355FIWPMLIHI Melanoma, Uterine Cancer 357 FLIQEIKTL HCC 358 FLTDYLNDL AML359 FMQDPMEVFV Melanoma, NHL, NSCLC, OC 360 HLIDTNKIQL NHL, NSCLC 361ILQEFESKL AML, NHL 362 ILTELGGFEV AML, PrC, BRCA, PC, CRC, HCC, HNSCC,Melanoma, NSCLC, OC, Esophageal Cancer, GC, RCC, SCLC, Urinary BladderCancer, Uterine Cancer 363 ITTEVVNELYV BRCA 365 LISPLLLPV SCLC, UterineCancer, HNSCC 367 NLWSLVAKV Uterine Cancer 368 QLQPTDALLCV UterineCancer 370 SIFASPESV Uterine Cancer, Gallbladder Cancer, Bile DuctCancer, AML, HNSCC 374 VLSVITEEL SCLC, HCC, PrC, Urinary Bladder Cancer,Uterine Cancer 375 VLWFKPVEL BRCA, OC, Uterine Cancer, GallbladderCancer, Bile Duct Cancer, HNSCC 377 VVDGTCVAV BRCA, Uterine Cancer,HNSCC 378 YILGKFFAL Melanoma, Esophageal Cancer 379 YLAELVTPIL HCC,BRCA, Uterine Cancer, AML, HNSCC 383 YLLREWVNL Melanoma, Uterine Cancer384 YMIGSEVGNYL SCLC, Uterine Cancer 385 YTIPLAIKL CRC NSCLC = non-smallcell lung cancer, SCLC = small cell lung cancer, RCC = kidney cancer,CRC = colon or rectum cancer, GC = stomach cancer, HCC = liver cancer,PC = pancreatic cancer, PrC = prostate cancer, BRCA = breast cancer, NHL= non-Hodgkin lymphoma, AML = acute myeloid leukemia, OC = ovariancancer, HNSCC = head and neck squamous cell carcinoma, CCC= cholangiocarcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 5, 7, 13, 29, 30, 35, 41, 44, 45, 51, 68, 93, 101,112, 115, 126, 130, 138, 141, 160, 161, 172, 187, 191, 197, 198, 204,212, 226, 228, 231, 232, 234, 258, 266, 268, 272, 279, 286, 289, 293,299, 303, 311, 313, 321, 323, 324, 327, 335, 336, 340, 343, 351, 353,355, 362, 365, 367, 368, 370, 374, 375, 377, 379, 383, and 384 forthe—in one preferred embodiment combined—treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 8, 11, 25, 42, 44, 67, 82, 84, 86, 91, 94, 114,136, 138, 145, 156, 161, 192, 202, 203, 204, 212, 216, 217, 218, 224,226, 237, 257, 261, 293, 294, 299, 313, 321, 323, 324, 334, 340, 353,358, 361, 362, 370, and 379 for the—in one preferred embodimentcombined—treatment of AML.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 8, 11, 14, 27, 38, 40, 48, 55, 59, 68, 84, 93, 94,114, 118, 124, 126, 128, 146, 159, 161, 162, 164, 176, 179, 188, 189,192, 200, 202, 208, 211, 212, 215, 216, 226, 228, 234, 235, 260, 264,272, 273, 274, 279, 280, 293, 298, 305, 311, 321, 339, 342, 343, 344,346, 348, 351, 353, 354, 362, 365, 370, 375, 377, and 379 for the—in onepreferred embodiment combined—treatment of HNSCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 11, 26, 37, 44, 49, 58, 83, 84, 113, 128, 173, 176,184, 201, 203, 226, 273, 279, 293, 317, 340, 348, 359, 360, and 361 forthe—in one preferred embodiment combined—treatment of NHL.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 11, 26, 45, 58, 84, 113, 136, 145, 173, 174, 176, 203,226, 279, 293, 326, 335, 348, 351, 359, 360, and 362 for the—in onepreferred embodiment combined—treatment of NSCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 11, 45, 68, 162, 173, 174, 226, 293, 351, 359, 362,and 375 for the—in one preferred embodiment combined—treatment of OC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 11, 35, 42, 59, 119, 126, 127, 152, 161, 172, 176,192, 226, 228, 239, 260, 280, 293, 324, 362, 365, 374, and 384 forthe—in one preferred embodiment combined—treatment of SCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 13, 14, 30, 68, 89, 108, 125, 198, 200, 204, 228, 235,242, 266, 269, 272, 280, 333, 353, 362, 363, 375, 377, and 379 forthe—in one preferred embodiment combined—treatment of BRCA.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 14, 30, 49, 90, 107, 108, 111, 126, 136, 147, 161,164, 173, 176, 181, 187, 190, 199, 208, 240, 274, 275, 280, 293, 340,349, 351, 355, 359, 362, 378, and 383 for the—in one preferredembodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 15, 82, 173, 211, 326, 362, and 378 for the—in onepreferred embodiment combined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 30, 136, 164, 173, 187, 266, 293, 298, 335, 351, 362,363, and 385 for the—in one preferred embodiment combined—treatment ofCRC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 44, 76, 84, 145, 173, 211, 293, 306, 326, 351, and 362for the—in one preferred embodiment combined—treatment of RCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 68, 94, 181, 226, 293, 335, 353, 370, and 375 forthe—in one preferred embodiment combined—treatment of gallbladder cancerand/or bile duct cancer and/or cholangiocarcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 82, 87, 162, 351, and 362 for the—in one preferredembodiment combined—treatment of PC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 84, 145, 173, 176, 258, 343, and 362 for the—in onepreferred embodiment combined—treatment of GC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 84, 126, 145, 173, 211, 239, 247, 349, 357, 362, 374,and 379 for the—in one preferred embodiment combined—treatment of HCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 136, 211, 221, 226, 293, 321, 335, 351, and 354 forthe—in one preferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 128, 172, 188, 192, 293, 305, 312, 321, 348, 362, and374 for the—in one preferred embodiment combined—treatment of urinarybladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 226, 321, 362, and 374 for the—in one preferredembodiment combined—treatment of PrC.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 385.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 385, preferably containing SEQ IDNo. 1 to SEQ ID No. 202, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are CLL, acute myelogenousleukemia, bile duct cancer, brain cancer, breast cancer, colorectalcarcinoma, esophageal cancer, gallbladder cancer, gastric cancer,hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkinlymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, renal cell cancer, small cell lung cancer, urinarybladder cancer and uterine cancer, and preferably CLL cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”,that can be used in the diagnosis of cancer, preferably CLL. The markercan be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin. The present invention also relatesto the use of these novel targets in the context of cancer treatment.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13, or 14 or longer,and in case of MHC class II peptides (elongated variants of the peptidesof the invention) they can be as long as 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

The term “full length polypeptide” means a complete protein (amino acidchain) or complete subunit (amino acid chain) of a multimeric protein.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans, there are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 −(1-Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles mightbe enriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype from Allele Populationallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 385, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 385. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 385, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according to SEQ ID NO: 2, 5,8, 62, and 53 Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 2 A L H R P DV Y L Variants V I A M M V M I M A A A V A I A A V V V V I V A T T V T IT A Q Q V Q I Q A SEQ ID NO 5 A L I F K I A S A Variants L I V M M L M IM V A A L A I A V V V L V I V V T T L T I T V Q Q L Q I Q V SEQ ID NO. 8A L A A S A L P A L V Variants I L A M M I M L M A A A I A L A A V V I VL V A T T I T L T A Q Q I Q L Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID 62 GI I D G S P R L Variants L V L I L L A M V M I M M A A V A I A A A V V VI V V A T V T I T T A Q V Q I Q Q A SEQ ID 153 S L L A E L H V LVariants V I A M V M I M M A A V A I A A A V V V I V V A T V T I T T A QV Q I Q Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 385.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 385 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 385and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄₁, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄₁, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNOB, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂,Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂), CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, Bale, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂), such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A-1V).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from CLL samples (N=17A*02-positive samples) with the fragmentation patterns of correspondingsynthetic reference peptides of identical sequences. Since the peptideswere directly identified as ligands of HLA molecules of primary tumors,these results provide direct evidence for the natural processing andpresentation of the identified peptides on primary cancer tissueobtained from 16 CLL patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from CLL tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary CLL samples confirming theirpresentation on primary CLL.

TUMAPs identified on multiple CLL and normal tissues were quantifiedusing ion-counting of label-free LC-MS data. The method assumes thatLC-MS signal areas of a peptide correlate with its abundance in thesample. All quantitative signals of a peptide in various LC-MSexperiments were normalized based on central tendency, averaged persample and merged into a bar plot, called presentation profile. Thepresentation profile consolidates different analysis methods likeprotein database search, spectral clustering, charge state deconvolution(decharging) and retention time alignment and normalization.

Furthermore, the discovery platform Technique XPRESIDENT® v2.x (see forexample PCT/EP2011/056056 and PCT/EP2015/079873) allows the directabsolute quantitation of MHC-, preferably HLA-restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured bynanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labelled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide:MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynanoLC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide-specificisolation efficiencies were calculated as an average from 10 spikeexperiments each measured as a triplicate (see Example 6 and Table 12).

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 3A-3F). Anadditional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably CLL that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanCLL samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy adipose tissue, adrenal gland, blood cells, blood vessel, bonemarrow, brain, breast, esophagus, eye, gallbladder, heart, kidney, largeintestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland,peritoneum, pituitary, pleura, salivary gland, skeletal muscle, skin,small intestine, spleen, stomach, thymus, thyroid gland, trachea,ureter, and urinary bladder cells or other normal tissue cells,demonstrating a high degree of tumor association of the source genes(see Example 2). Moreover, the peptides themselves are stronglyover-presented on tumor tissue—“tumor tissue” in relation to thisinvention shall mean a sample from a patient suffering from CLL, but noton normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. CLL cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule. The presentdescription also relates to nucleic acids, vectors and host cells forexpressing TCRs and peptides of the present description; and methods ofusing the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A2-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are upregulated or overexpressed (socalled self-antigens) will not necessarily induce a functional immuneresponse against the tumor: T-cells expressing TCRs that are highlyreactive to these antigens will have been negatively selected within thethymus in a process known as central tolerance, meaning that onlyT-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to peptides canbe enhanced by methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 385, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment, the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonal®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonal®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment, a scaffold is ableto activate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry. Each scaffold can be conjugatedwith a second active molecule such as for example IL-21, anti-CD3,anti-CD28. For further information on polypeptide scaffolds see forexample the background section of WO 2014/071978A1 and the referencescited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed, aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 385,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 385, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:385 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 385, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 385.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of CLL.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing or expressing said peptide containing SEQID NO: 1 to SEQ ID NO: 385 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are CLL cells or other solid orhematological tumor cells such as acute myelogenous leukemia, bile ductcancer, brain cancer, breast cancer, colorectal carcinoma, esophagealcancer, gallbladder cancer, gastric cancer, hepatocellular cancer,Merkel cell carcinoma, melanoma, non-Hodgkin lymphoma, non-small celllung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renalcell cancer, small cell lung cancer, urinary bladder cancer and uterinecancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of CLL. The present invention also relates to the use of thesenovel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a CLL marker (poly)peptide,delivery of a toxin to a CLL cell expressing a cancer marker gene at anincreased level, and/or inhibiting the activity of a CLL markerpolypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length CLL marker polypeptides or fragments thereof maybe used to generate the antibodies of the invention. A polypeptide to beused for generating an antibody of the invention may be partially orfully purified from a natural source, or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 385polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the CLL marker polypeptide used togenerate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating CLL, the efficacyof the therapeutic antibody can be assessed in various ways well knownto the skilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 385, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al. (Porta et al., 1994) which describes the development ofcowpea mosaic virus as a high-yielding system for the presentation offoreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 385.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from CLL, themedicament of the invention is preferably used to treat CLL.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of CLLpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several CLLtissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPS in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, CLL samples from patients andblood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (CLL)compared with a range of normal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromCLL patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory, an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In one aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from CLL cells and since it was determined that thesepeptides are not or at lower levels present in normal tissues, thesepeptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for CLL. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A to 1V show the overpresentation of various peptides in normaltissues (white bars) and CLL (black bars). FIG. 1A) Gene: IGHM, Peptide:ALHRPDVYL (SEQ ID NO.: 2). Tissues from left to right: 2 adiposetissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bonemarrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymphnodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7salivary glands, 5 skeletal muscles, 6 skins, 4 small intestines, 9spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3ureters, 6 urinary bladders, 2 uteri, 6 esophagi, 17 chronic lymphocyticleukemia samples. The peptide has additionally been detected on 2/21non-Hodgkin lymphoma samples. FIG. 1B) Gene: IGHM, Peptide: VIAELPPKV(SEQ ID NO.: 3). Tissues from left to right: 2 adipose tissues, 3adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts, 14kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymph nodes, 7nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1 peritoneum, 5pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivaryglands, 5 skeletal muscles, 6 skins, 4 small intestines, 9 spleens, 5stomachs, 6 testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6urinary bladders, 2 uteri, 6 esophagi, 17 chronic lymphocytic leukemiasamples. The peptide has additionally been detected on 5/21 non-Hodgkinlymphoma samples and 1/90 lung cancers. FIG. 1C), Gene: IGHM, Peptide:VIAELPPKVSV (SEQ ID No.: 4), Tissues from left to right: 2 adiposetissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bonemarrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymphnodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7salivary glands, 5 skeletal muscles, 6 skins, 4 small intestines, 9spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3ureters, 6 urinary bladders, 2 uteri, 6 esophagi, 17 chronic lymphocyticleukemia samples. FIG. 1D), Gene: NUP210, Peptide: RLYEITIEV (SEQ IDNo.: 121). Samples from left to right: 1 PBMC culture, 1 benignprostate, 8 normal tissues (3 lungs, 4 spleens, 1 trachea), 49 cancertissues (2 brain cancers, 2 breast cancers, 1 colon cancer, 13leukocytic leukemia cancers, 13 lung cancers, 8 lymph node cancers, 1myeloid cells cancer, 4 ovarian cancers, 2 skin cancers, 1 urinarybladder cancer, 2 uterus cancers). Discrepancies regarding the list oftumor types between FIG. 1D and table 4 may be due to the more stringentselection criteria applied in table 4 (for details please refer to table4). The normal tissue panel and the cancer cell lines and xenograftstested were the same as in FIGS. 1A-1C. FIG. 1E), Gene: COBRA1, Peptide:ALLRLLPGL (SEQ ID No.: 14). Samples from left to right: 1 cell line, 11cancer tissues (1 breast cancer, 1 colon cancer, 1 head and neck cancer,2 leukocytic leukemia cancers, 1 liver cancer, 1 lung cancer, 2 lymphnode cancers, 1 myeloid cell cancer, 1 skin cancer). FIG. 1F), Gene:KIAA0226L, Peptide: GIIDGSPRL (SEQ ID No.: 62). Samples from left toright: 2 normal tissues (1 lymph node, 1 spleen), 18 cancer tissues (2colon cancers, 9 leukocytic leukemia cancers, 6 lymph node cancers, 1rectum cancer). FIG. 1G), Gene: DDX3X, Peptide: GLDQQFAGLDL (SEQ ID No.:68). Samples from left to right: 1 cell line, 2 primary cultures, 21cancer tissues (1 bile duct cancer, 1 brain cancer, 1 breast cancer, 1head and neck cancer, 2 kidney cancers, 3 leukocytic leukemia cancers, 2liver cancers, 2 lung cancers, 2 lymph node cancers, 3 ovarian cancers,1 rectum cancer, 1 skin cancer, 1 uterus cancer). FIG. 1H), Gene: LIG1,Peptide: KTLDVDATYEI (SEQ ID No.: 112). Samples from left to right: 2cell lines, 1 primary culture, 13 cancer tissues (1 esophageal cancer, 2leukocytic leukemia cancers, 1 liver cancer, 3 lung cancers, 2 lymphnode cancers, 2 ovarian cancers, 1 skin cancer, 1 uterus cancer). FIG.1I), Gene: SMCHD1, Peptide: SIIEGPIIKL (SEQ ID No.: 146). Samples fromleft to right: 14 cancer tissues (1 head and neck cancer, 3 leukocyticleukemia cancers, 6 lung cancers, 1 lymph node cancer, 1 ovarian cancer,2 urinary bladder cancers). FIG. 1J), Gene: MMS22L, Peptide: SILETVATL(SEQ ID No.: 147). Samples from left to right: 1 primary culture, 11cancer tissues (1 colon cancer, 4 leukocytic leukemia cancers, 1 lungcancer, 3 lymph node cancers, 1 myeloid cell cancer, 1 skin cancer).FIG. 1K), Gene: FCRL3, Peptide: SLLAELHVL (SEQ ID No.: 153). Samplesfrom left to right: 1 cell line, 9 normal tissues (1 lymph node, 1rectum, 7 spleens), 27 cancer tissues (1 breast cancer, 1 kidney cancer,10 leukocytic leukemia cancers, 14 lymph node cancers, 1 skin cancer).FIG. 1L), Gene: FAM126A, Peptide: SLQEEKLIYV (SEQ ID No.: 159). Samplesfrom left to right: 2 cell lines, 23 cancer tissues (5 brain cancers, 1breast cancer, 1 esophageal cancer, 3 head and neck cancers, 2leukocytic leukemia cancers, 5 lung cancers, 3 lymph node cancers, 1skin cancer, 2 urinary bladder cancers). FIG. 1M), Gene: HNRNPC,Peptide: SVHKGFAFV (SEQ ID No.: 164). Samples from left to right: 10cell lines, 1 primary culture, 1 normal tissue (1 adrenal gland), 36cancer tissues (12 brain cancers, 1 colon cancer, 2 head and neckcancers, 2 kidney cancers, 3 leukocytic leukemia cancers, 1 livercancer, 5 lung cancers, 1 myeloid cell cancer, 1 ovarian cancer, 1rectum cancer, 1 skin cancer, 2 stomach cancers, 2 urinary bladdercancers, 2 uterus cancers). FIG. 1N), Gene: RASGRF1, Peptide: TLDTSKLYV(SEQ ID No.: 167). Samples from left to right: 13 cancer tissues (1brain cancer, 8 leukocytic leukemia cancers, 4 lymph node cancers). FIG.1O), Gene: RASGRF1, Peptide: YLLDQSFVM (SEQ ID No.: 168). Samples fromleft to right: 16 cancer tissues (10 leukocytic leukemia cancers, 6lymph node cancers). FIG. 1P), Gene: RNF213, Peptide: YIQEYLTLL (SEQ IDNo.: 192). Samples from left to right: 2 cell lines, 18 cancer tissues(1 colon cancer, 1 head and neck cancer, 1 kidney cancer, 4 leukocyticleukemia cancers, 5 lung cancers, 3 lymph node cancers, 1 myeloid cellcancer, 1 stomach cancer, 1 urinary bladder cancer). FIG. 1Q), Gene:DCK, Peptide: YLQEVPILTL (SEQ ID No.: 197). Samples from left to right:11 cancer tissues (5 leukocytic leukemia cancers, 4 lymph node cancers,1 skin cancer, 1 uterus cancer). FIG. 1R), Gene: EIF3H, Peptide:YMFEEVPIVI (SEQ ID No.: 200). Samples from left to right: 4 cell lines,2 primary cultures, 34 cancer tissues (1 bile duct cancer, 2 breastcancers, 1 colon cancer, 1 esophageal cancer, 1 gallbladder cancer, 5head and neck cancers, 2 leukocytic leukemia cancers, 2 liver cancers, 9lung cancers, 4 lymph node cancers, 2 ovarian cancers, 1 rectum cancer,2 skin cancers, 1 urinary bladder cancer). FIG. 1S), Gene: UBLCP1,Peptide: LIDVKPLGV (SEQ ID No.: 211). Samples from left to right: 3 celllines, 23 cancer tissues (5 brain cancers, 1 breast cancer, 1 esophagealcancer, 1 head and neck cancer, 1 kidney cancer, 3 leukocytic leukemiacancers, 2 liver cancers, 2 lung cancers, 4 lymph node cancers, 1ovarian cancer, 1 prostate cancer, 1 skin cancer). FIG. 1T), Gene:TMCO6, Peptide: QLLPVSNVVSV (SEQ ID No.: 256). Samples from left toright: 2 primary cultures, 13 cancer tissues (2 head and neck cancers, 3leukocytic leukemia cancers, 1 liver cancer, 2 lung cancers, 4 lymphnode cancers, 1 ovarian cancer). FIG. 1U), Gene: MYO1G, Peptide:LLYNSTDPTL (SEQ ID No.: 312). Samples from left to right: 1 cell line, 2primary cultures, 14 cancer tissues (1 colon cancer, 4 leukocyticleukemia cancers, 2 lung cancers, 5 lymph node cancers, 1 ovariancancer, 1 urinary bladder cancer). FIG. 1V), Gene: PIM1, Peptide:VLLPQETAEIHL (SEQ ID No.: 328). Samples from left to right: 3 primarycultures, 27 cancer tissues (1 breast cancer, 1 colon cancer, 5leukocytic leukemia cancers, 5 lung cancers, 7 lymph node cancers, 2ovarian cancers, 2 skin cancers, 1 stomach cancer, 3 urinary bladdercancers).

FIGS. 2A to 2D show exemplary expression profiles of source genes of thepresent invention that are highly over-expressed or exclusivelyexpressed in CLL in a panel of normal tissues (white bars) and 17 CLLsamples (black bars). Tissues from left to right: 6 arteries, 1 bloodcells, 1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose tissue, 1adrenal gland, 6 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 5 pancreases, 2 pituitaryglands, 1 rectum, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 smallintestine, 1 spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinarybladder, 1 breast, 5 ovaries, 3 placentas, 1 prostate, 1 testis, 1thymus, 1 uterus, 10 chronic lymphocytic leukemia samples. FIG. 2A) Genesymbol: FCER2; FIG. 2B) Gene symbol: KIAA0226L; FIG. 2C) Gene symbol:PAX5; FIG. 2D) Gene symbol: CLEC17A.

FIGS. 3A-3F show exemplary immunogenicity data: flow cytometry resultsafter peptide-specific multimer staining. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex withSeqID No 1 peptide (FIG. 3C, left panel), SeqID No 5 peptide (FIG. 3Dleft panel), SeqID No 32 peptide (FIG. 3E, left panel) and SeqID No 220peptide (FIG. 3F, left panel), respectively. After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*02/SeqID No 1 (FIG. 3C), A*02/SeqID No 5 (D),A*02/SeqID No 32 (FIG. 3E) or A*02/SeqID No 220 (FIG. 3F). Right panels(FIGS. 3C, 3D, 3E and 3F) show control staining of cells stimulated withirrelevant A*02/peptide complexes. Viable singlet cells were gated forCD8+ lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1 Identification and Quantitation of Tumor AssociatedPeptides Presented on the Cell Surface Tissue Samples

Patients' tumor tissues were obtained from: ProteoGenex Inc. (CulverCity, Calif., USA); University Hospital Bonn (Bonn, Germany); UniversityHospital Tübingen (Tübingen, Germany).

Normal tissues were obtained from Asterand (Detroit, Mich., USA &Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University ofMedicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif.,USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Munich (Munich, Germany); UniversityHospital Tübingen (Tübingen, Germany).

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide, a presentation profile was calculatedshowing the mean sample presentation as well as replicate variations.The profiles juxtapose CLL samples to a baseline of normal tissuesamples. Presentation profiles of exemplary over-presented peptides areshown in FIGS. 1A-1V. Presentation scores for exemplary peptides areshown in Table 8.

TABLE 8 Presentation scores. The table lists peptides that are veryhighly over-presented on tumors compared to a panel of normal tissues(+++), highly over-presented on tumors compared to a panel of normaltissues (++) or over-presented on tumors compared to a panel of normaltissues (+). The panel of normal tissues considered relevant forcomparison with tumors consisted of: adipose tissue, adrenal gland,blood cells, blood vessel, bone marrow, brain, breast, esophagus, eye,gallbladder, heart, kidney, large intestine, liver, lung, lymph node,nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura,salivary gland, skeletal muscle, skin, small intestine, spleen, stomach,thymus, thyroid gland, trachea, ureter, urinary bladder. SEQ ID PeptideNo. Sequence Presentation 1 AIPPSFASIFL +++ 2 ALHRPDVYL +++ 3 VIAELPPKV+++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 6 ALDTLEDDMTI +++ 7 ALLERTGYTL +++8 ALAASALPALV +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 11 ALFTFJPLTV +++ 12ALGEDEITL +++ 13 VVDGMPPGV ++ 14 ALLRLLPGL + 15 ALPEVSVEA + 16ALPGGAAVAAV ++ 17 ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV + 20ALVDPGPDFVV + 21 ALWAGLLTL +++ 22 ALWDPVIEL +++ 23 ALYLTEVFL +++ 24AMAGDVVYA +++ 25 ATYSGLESQSV + 27 AVLGLVWLL ++ 28 AVLHLLLSV +++ 29AVLQAVTAV +++ 30 ELLEGSEIYL + 31 ELMHGVAGV ++ 32 FIDKFTPPV +++ 33FIINSSNIFL +++ 35 FILPSSLYL +++ 36 FILTHVDQL +++ 37 FIMEGGAMVL +++ 39FLDALLTDV +++ 40 FLDEDDMSL + 41 FLDPSLDPLL +++ 43 FLLGPEALSFA +++ 45FLPELPADLEA ++ 47 FLSDQPEPYL ++ 48 FLSPQQPPLL +++ 49 FLTDLFAQL + 50FLFEPVVKAFL +++ 51 FLVEAPHDWDL ++ 52 FLVETGFLHV +++ 53 FLWQHVELVL +++ 54FLYPFPLALF +++ 56 FVFEAPYTL +++ 57 GLSEISLRL +++ 58 YIQQGIFSV ++ 59FVFGDENGTVSL +++ 60 FVLDHEDGLNL ++ 61 FVYFIVREV +++ 62 GIIDGSPRL + 63SLAHVAGCEL +++ 64 KLLESVASA ++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 68GLDQQFAGLDL +++ 69 GLHQREIFL +++ 70 FVPDTPVGV ++ 71 GLKHDIARV +++ 72GLLDAGKMYV +++ 73 GLLEVISALQL +++ 74 GLLRASFLL +++ 76 GLLRIIPYL ++ 77GLLRLTWFL ++ 78 GLPSFLTEV +++ 79 GLQAKIQEA +++ 80 VLIEDELEEL +++ 81WLVGQEFEL +++ 82 GLQSGVDIGV + 83 GQGEVLVYV + 86 HLYPGAVTI +++ 88ILDEIGADVQA +++ 89 ILDFGTFQL +++ 90 VIADLGLIRV +++ 92 ILEPLNPLL +++ 93ILFNTQINI +++ 94 ILFPLRFTL +++ 95 ILGYMAHEHKV +++ 96 ILIDKTSFV + 97LLFATQITL + 98 SLIKYFLFV +++ 99 ILIFHSVAL +++ 100 ILNNEVFAI + 101ILVVIEPLL +++ 102 IQDRAVPSL +++ 103 KLGGTPAPA ++ 104 KLILLDTPLFL +++ 105KLMNDIADI +++ 106 FMASHLDYL +++ 107 ILYNLYDLL ++ 108 VIYTLIHYI + 109KLWEGLTELV ++ 112 KTLDVDATYEI +++ 113 KVPAEEVLVAV + 114 LIPEGPPQV +++115 LLFDKLYLL +++ 116 LLIGATMQV +++ 117 LLILENILL ++ 118 RLLILENILL +++119 VLPAEFFEV +++ 121 RLYEITIEV ++ 122 LLIPVVPGV ++ 123 LLLAEAELLTL +++124 LLLEETEKQAV ++ 125 LLLEIGEVGKLFV ++ 126 LLPEGGITAI +++ 127 LLPTAPTTV+++ 128 LLSEEEYHL +++ 129 LLVGTLDVV + 130 LLVLIPVYL +++ 131 LQALEVLKI+++ 132 LVYEAIIMV +++ 133 YLLSGDISEA +++ 134 MLLEHGITLV +++ 135MTAGFSTIAGSV +++ 137 NLIKTVIKL + 138 NLLDIDAPVTV ++ 139 NLTDVVEKL +++140 QIAELPATSV +++ 141 QILSEIVEA ++ 142 QLDEPAPQV +++ 144 QLPPFPREL +++145 SALDTITTV +++ 146 SIIEGPIIKL +++ 147 SILETVATL +++ 148 SIVASLITV +++149 SLDNGGYYI +++ 153 SLLAELHVL ++ 154 SLLAELHVLTV ++ 155 SLMLEVPAL +++157 SLNIRDFTM +++ 160 SLSFLVPSL +++ 162 SMKDDLENV +++ 163 SQLDISEPYKV +164 SVHKGFAFV +++ 165 TLDDDLDTV +++ 166 TLDPNQVSL +++ 167 TLDTSKLYV +++168 YLLDQSFVM +++ 169 TLLLGLTEV +++ 170 TLTFRVETV +++ 171 TLVPPAALISI+++ 172 TLYDMLASI +++ 174 VLAELPIIVV +++ 175 FTVPRVVAV ++ 176VLAEQNIIPSA +++ 177 VLDDRELLL +++ 178 VLFFNVQEV +++ 179 VLLGLEMTL +++181 VLLSIPFVSV + 182 VLLSVPGPPV +++ 185 VMDDQRDLI + 186 VMDPTKILI +++187 VMDTHLVNI +++ 189 VMLEMTPEL +++ 190 VVMGTVPRL +++ 191 YIFDGSDGGV +++192 YIQEYLTLL +++ 193 YLDLSNNRL +++ 194 YLDNVLAEL + 195 YLGGFALSV +++197 YLQEVPILTL +++ 198 YLTFLPAEV +++ 199 YLVELSSLL +++ 200 YMFEEVPIVI+++ 201 YQLELHGIEL +++ 202 YVDDVFLRV +++ 204 GLLQINDKIAL ++ 205GLSQANFTL + 206 HMQDVRVLL +++ 208 ILLKTEGINL ++ 209 ILQAELPSL ++ 210KLLVQDFFL +++ 211 LIDVKPLGV +++ 212 NIIEAINELLV ++ 213 RLLYQLVFL ++ 214RLQELTEKL + 215 VMQDIVYKL + 217 ALDEPPYLTV + 218 ALGEEWKGYVV + 219ALLNLLESA ++ 221 ALVSTIIMV + 225 ILQERELLPV + 226 AMNISVPQV + 227FLAEASVMTQL ++ 228 FLGGLSPGV ++ 229 FLLNLQNCHL ++ 231 FLYIRQLAI + 232FMHQIIDQV + 234 GLDDAEYAL + 235 GLDDLLLFL + 236 GLLESGRHYL +++ 237GLQENLDVVV + 238 GLVETELQL + 240 ILARDILEI ++ 241 ILGDILLKV + 242ILLGIQELL ++ 243 ILPTLEKELFL + 244 ILQALAVHL ++ 245 KIMDYSLLLGV + 248KTVEPPISQV + 249 LLPTGVFQV + 251 LLYDNVPGA + 254 QLIPKLIFL +++ 255YLFEEAISM + 258 RLDYITAEI ++ 259 RLLDEQFAVL + 260 SLDDVEGMSV ++ 261SLVEAQGWLV ++ 263 SQWEDIHVV ++ 264 TILDYINVV +++ 266 TLLDQLDTQL + 267TLLDWQDSL + 268 TLLQVFHLL ++ 270 TVLPVPPLSV +++ 271 VIRNIVEAA ++ 273VLGEYSYLL + 275 VLLFIEHSV + 276 VLNDGAPNV + 277 VMILKLPFL + 278YLDDLLPKL ++ 280 YTLDSLYWSV + 281 NLLDDRGMTAL +++ 282 LLRDGIELV +++ 283ILQPMDIHV +++ 284 LLSAAEPVPA +++ 286 FLLEDLSQKL +++ 287 FLWEEKFNSL +++289 ILEEQPMDMLL +++ 290 LANPHELSL +++ 291 ILLNEDDLVTI +++ 292AAALIIHHV + 294 ALLDQLHTLL + 297 FLVEPQEDTRL ++ 298 IILPVEVEV +++ 299ILEENIPVL ++ 300 ILLNPAYDVYL ++ 302 ILQDLTFVHL + 304 LAIVPVNTL +++ 305LLFPQIEGIKI + 307 LLLTKPTEA ++ 308 SLYDVSRMYV +++ 309 ILYGTQFVL + 310LLSTLHLLV + 311 LLVDVEPKV + 312 LLYNSTDPTL +++ 314 LMKDCEAEV ++ 316MLLEHGITL +++ 317 NLLAHIWAL + 318 NLQVTQPTV +++ 320 TIAPVTVAV + 322SLASIHVPL +++ 323 SLDLFNCEV +++ 327 VLIKWFPEV +++ 328 VLLPQETAEIHL ++329 VLMDGSVKL + 330 VLMWEIYSL ++ 331 VLWELAHLPTL +++ 332 VMIQHVENL +++334 YLLEEKIASL +++ 336 YMAVTTQEV +++ 337 YMYEKESEL ++ 338 FLDMTNWNL +339 GLWGTVVNI + 340 KLLEEICNL ++ 341 LLAELPASVHA ++ 342 SLITPLQAV +++343 TLLEALDCI ++ 344 VLAFENPQV ++ 345 YLIEPDVELQRI + 346 VLVQVSPSL +++347 YLGPVSPSL + 349 ALATHILSL ++ 350 ALEDRVWEL + 351 ALSEKLARL + 352ALVFELHYV + 353 ATPMPTPSV ++ 354 FIMDDPAGNSYL + 355 FIWPMLIHI +++ 356FLHDHQAEL + 357 FLIQEIKTL ++ 358 FLTDYLNDL + 359 FMQDPMEVFV ++ 360HLIDTNKIQL + 362 ILTELGGFEV + 363 ITTEVVNELYV + 364 KMDWIFHTI + 365LISPLLLPV ++ 367 NLWSLVAKV ++ 368 QLQPTDALLCV + 371 SLADDSVLERL + 372SLFGPLPGPGPALV + 374 VLSVITEEL ++ 375 VLWFKPVEL ++ 377 VVDGTCVAV + 378YILGKFFAL + 379 YLAELVTPIL + 380 YLDRKLLTL + 382 YLLPLLQRL +++ 383YLLREWVNL +++ 384 YMIGSEVGNYL + 385 YTIPLAIKL +++

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); BioServe (Beltsville, Md., USA); Geneticist Inc.(Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples,Italy); ProteoGenex Inc. (Culver City, Calif., USA); University HospitalHeidelberg (Heidelberg, Germany).

Total RNA from tumor tissues for RNASeq experiments was obtained from:Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn,Germany)

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (Illumina Inc,San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolarly and sequenced on the IlluminaHiSeq 2500 sequencer according to the manufacturer's instructions,generating 50 bp single end reads. Processed reads are mapped to thehuman genome (GRCh38) using the STAR software. Expression data areprovided on transcript level as RPKM (Reads Per Kilobase per Millionmapped reads, generated by the software Cufflinks) and on exon level(total reads, generated by the software Bedtools), based on annotationsof the ensembl sequence database (Ensembl77). Exon reads are normalizedfor exon length and alignment size to obtain RPKM values. Exemplaryexpression profiles of source genes of the present invention that arehighly over-expressed or exclusively expressed in CLL are shown in FIGS.3A-3F. Expression scores for further exemplary genes are shown in Table9.

TABLE 9 Expression scores. The table lists peptides from genes that arevery highly over-expressed in tumors compared to a panel of normaltissues (+++), highly over-expressed in tumors compared to a panel ofnormal tissues (++) or over-expressed in tumors compared to a panel ofnormal tissues (+). The baseline for this score was calculated frommeasurements of the following relevant normal tissues: adipose tissue,adrenal gland, artery, blood cells, bone marrow, brain, cartilage,colon, esophagus, eye, gallbladder, heart, kidney, liver, lung, lymphnode, pancreas, pituitary, rectum, salivary gland, skeletal muscle,skin, small intestine, spleen, stomach, thyroid gland, trachea, urinarybladder, and vein. In case expression data for several samples of thesame tissue type were available, the arithmetic mean of all respectivesamples was used for the calculation. Gene SEQ ID No Sequence Expression1 AIPPSFASIFL ++ 2 ALHRPDVYL ++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5ALIFKIASA +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 17 ALTKTNLQL +++ 18LLGEFSIKM +++ 19 QVMEKLAAV +++ 21 ALWAGLLTL +++ 24 AMAGDVVYA +++ 31ELMHGVAGV +++ 46 YIIDSAQAV ++ 62 GIIDGSPRL +++ 63 SLAHVAGCEL +++ 64KLLESVASA +++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++ 71 GLKHDIARV +++ 74GLLRASFLL ++ 75 GLSIFAQDLRL ++ 85 HLMLHTAAL + 86 HLYPGAVTI +++ 91ILDLNTYNV + 96 ILIDKTSFV +++ 97 LLFATQITL +++ 98 SLIKYFLFV +++ 105KLMNDIADI ++ 106 FMASHLDYL + 107 ILYNLYDLL ++ 108 VIYTLIHYI + 113KVPAEEVLVAV ++ 116 LLIGATMQV + 117 LLILENILL ++ 118 RLLILENILL ++ 119VLPAEFFEV ++ 120 AIDAALTSV ++ 121 RLYEITIEV ++ 127 LLPTAPTTV + 137NLIKTVIKL + 146 SIIEGPIIKL +++ 149 SLDNGGYYI +++ 150 SLFDQPLSII +++ 153SLLAELHVL +++ 154 SLLAELHVLTV +++ 156 SLNIGDVQL +++ 163 SQLDISEPYKV +++177 VLDDRELLL ++ 189 VMLEMTPEL +++ 193 YLDLSNNRL + 195 YLGGFALSV ++ 205GLSQANFTL +++ 206 HMQDVRVLL +++ 213 RLLYQLVFL +++ 220 ALPEILFAKV ++ 221ALVSTIIMV + 223 ALWVSQPPEI ++ 235 GLDDLLLFL + 251 LLYDNVPGA +++ 254QLIPKLIFL +++ 255 YLFEEAISM +++ 257 RIINGIIISV +++ 262 SLWNAGTSV ++ 275VLLFIEHSV ++ 284 LLSAAEPVPA +++ 285 GVATAGCVNEV +++ 286 FLLEDLSQKL ++294 ALLDQLHTLL + 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 301 AASPIITLV + 307LLLTKPTEA ++ 308 SLYDVSRMYV + 309 ILYGTQFVL ++ 312 LLYNSTDPTL + 319QVIPQLQTV ++ 320 TIAPVTVAV ++ 330 VLMWEIYSL ++ 332 VMIQHVENL +++ 337YMYEKESEL +++ 352 ALVFELHYV +++ 356 FLHDHQAEL + 360 HLIDTNKIQL + 378YILGKFFAL + 380 YLDRKLLTL ++

Example 3 In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 523) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.524), respectively.

800,000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+ CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for CLL Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for twopeptides of the invention are shown in FIGS. 3A-3F together withcorresponding negative controls. Results for two peptides from theinvention are summarized in Table 10A, others in Table 10B.

TABLE 10A in vitro immunogenicity of HLA class I peptides of theinvention Seq ID Sequence wells 427 LLYDAVHIV ++ 521 YLYGQTTTYL ++Exemplary results of in vitro immunogenicity experiments conducted bythe applicant for the peptides of the invention. <20% = +; 20%-49% = ++;50%-69% = +++; >=70% = ++++

TABLE 10B In vitro immunogenicity of additional HLA class I peptides ofthe invention SEQ ID NO: Sequence Wells positive [%] 1 AIPPSFASIFL “+++”2 ALHRPDVYL “+” 5 ALIFKIASA “+++” 17 ALTKTNLQL “+” 26 AVLLVLPLV “+” 32FIDKFTPPV “++++” 49 FLTDLFAQL “++” 54 FLYPFPLALF “+” 56 FVFEAPYTL “++”57 GLSEISLRL “+” 69 GLHQREIFL “+” 81 WLVGQEFEL “+” 86 HLYPGAVTI “+” 96ILIDKTSFV “+” 100 ILNNEVFAI “++” 101 ILVVIEPLL “+” 102 IQDRAVPSL “+” 112KTLDVDATYEI “+” 114 LIPEGPPQV “+” 154 SLLAELHVLTV “+” 192 YIQEYLTLL “+”197 YLQEVPILTL “+” 200 YMFEEVPIVI “+” 201 YQLELHGIEL “+” 202 YVDDVFLRV“++” 220 ALPEILFAKV “++” 240 ILARDILEI “+” 281 NLLDDRGMTAL “+” 282LLRDGIELV “++” 307 LLLTKPTEA “+” 320 TIAPVTVAV “+” 322 SLASIHVPL “+” 323SLDLFNCEV “+” 329 VLMDGSVKL “+++” 332 VMIQHVENL “+” 380 YLDRKLLTL “++++”Exemplary results of in vitro immunogenicity experiments conducted bythe applicant for HLA-A*02 restricted peptides of the invention. Resultsof in vitro immunogenicity experiments are indicated. Percentage ofpositive wells and donors (among evaluable) are summarized as indicated<20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++

Example 4 Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5 MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH2SO4. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. SEQID Sequence Peptide Exchange 1AIPPSFASIFL “+++” 2 ALHRPDVYL “+++” 3 VIAELPPKV “++” 4 VIAELPPKVSV “+++”5 ALIFKIASA “+++” 6 ALDTLEDDMTI “+++” 7 ALLERTGYTL “+++” 8 ALAASALPALV“+++” 9 ALCDTLITV “+++” 10 FVYGESVEL “+++” 11 ALFTFJPLTV “+++” 12ALGEDEITL “+++” 13 VVDGMPPGV “+++” 14 ALLRLLPGL “+++” 15 ALPEVSVEA “+++”16 ALPGGAAVAAV “++++” 17 ALTKTNLQL “+++” 18 LLGEFSIKM “+++” 19 QVMEKLAAV“+++” 20 ALVDPGPDFVV “+++” 21 ALWAGLLTL “+++” 22 ALWDPVIEL “+++” 23ALYLTEVFL “++++” 24 AMAGDVVYA “+++” 25 ATYSGLESQSV “++” 26 AVLLVLPLV“+++” 27 AVLGLVWLL “+++” 28 AVLHLLLSV “++” 29 AVLQAVTAV “+++” 30ELLEGSEIYL “+++” 31 ELMHGVAGV “+++” 32 FIDKFTPPV “++++” 34 YLPYIFPNI“+++” 35 FILPSSLYL “+++” 36 FILTHVDQL “++++” 37 FIMEGGAMVL “++++” 38FIMEGGAMV “+++” 39 FLDALLTDV “+++” 40 FLDEDDMSL “++” 41 FLDPSLDPLL “+++”42 FLEEGGVVTV “+++” 43 FLLGPEALSFA “++++” 44 FLLSINDFL “+++” 45FLPELPADLEA “+++” 46 YIIDSAQAV “+++” 47 FLSDQPEPYL “++” 48 FLSPQQPPLL“+++” 49 FLTDLFAQL “++++” 50 FLFEPVVKAFL “+++” 51 FLVEAPHDWDL “++++” 52FLVETGFLHV “++++” 53 FLWQHVELVL “+++” 54 FLYPFPLALF “+++” 55 FMEPTLLML“+++” 56 FVFEAPYTL “+++” 57 GLSEISLRL “++++” 58 YIQQGIFSV “+++” 59FVFGDENGTVSL “+++” 60 FVLDHEDGLNL “+++” 61 FVYFIVREV “+++” 62 GIIDGSPRL“+++” 63 SLAHVAGCEL “+++” 64 KLLESVASA “+++” 65 GLDDMKANL “++” 66SLAGGLDDMKA “+++” 67 GLDDVTVEV “++” 68 GLDQQFAGLDL “+” 69 GLHQREIFL“+++” 70 FVPDTPVGV “+++” 71 GLKHDIARV “+++” 72 GLLDAGKMYV “+++” 73GLLEVISALQL “+++” 74 GLLRASFLL “++” 75 GLSIFAQDLRL “++” 76 GLLRIIPYL“+++” 78 GLPSFLTEV “+++” 79 GLQAKIQEA “+++” 80 VLIEDELEEL “++” 81WLVGQEFEL “+++” 82 GLQSGVDIGV “+++” 83 GQGEVLVYV “+++” 84 GVMDVNTAL “++”85 HLMLHTAAL “++++” 86 HLYPGAVTI “+++” 87 HQIEAVDGEEL “++” 88ILDEIGADVQA “+++” 89 ILDFGTFQL “+++” 90 VIADLGLIRV “+++” 91 ILDLNTYNV“+++” 92 ILEPLNPLL “+++” 93 ILFNTQINI “+++” 94 ILFPLRFTL “+++” 95ILGYMAHEHKV “++” 96 ILIDKTSFV “+++” 97 LLFATQITL “++” 98 SLIKYFLFV “+”99 ILIFHSVAL “+++” 100 ILNNEVFAI “+++” 101 ILVVIEPLL “++++” 102IQDRAVPSL “++” 103 KLGGTPAPA “++” 104 KLILLDTPLFL “+++” 105 KLMNDIADI“++” 106 FMASHLDYL “+++” 107 ILYNLYDLL “+++” 108 VIYTLIHYI “+++” 109KLWEGLTELV “+++” 110 LLFDHLEPMEL “+++” 111 KLWNVAAPLYL “+++” 112KTLDVDATYEI “+++” 113 KVPAEEVLVAV “+++” 114 LIPEGPPQV “+++” 115LLFDKLYLL “+++” 116 LLIGATMQV “+++” 117 LLILENILL “+++” 118 RLLILENILL“+++” 119 VLPAEFFEV “+++” 120 AIDAALTSV “+++” 121 RLYEITIEV “++++” 122LLIPVVPGV “+++” 123 LLLAEAELLTL “++” 124 LLLEETEKQAV “+++” 125LLLEIGEVGKLFV “+++” 126 LLPEGGITAI “+++” 127 LLPTAPTTV “+++” 128LLSEEEYHL “+++” 130 LLVLIPVYL “+” 131 LQALEVLKI “+” 132 LVYEAIIMV “+++”133 YLLSGDISEA “+++” 134 MLLEHGITLV “++++” 135 MTAGFSTIAGSV “+” 136NLDKLWTLV “++” 137 NLIKTVIKL “+++” 138 NLLDIDAPVTV “+++” 139 NLTDVVEKL“+++” 140 QIAELPATSV “+++” 141 QILSEIVEA “++” 142 QLDEPAPQV “++” 143QLLDTYFTL “+++” 145 SALDTITTV “+++” 146 SIIEGPIIKL “+++” 147 SILETVATL“+++” 148 SIVASLITV “+++” 149 SLDNGGYYI “+++” 150 SLFDQPLSII “+++” 151SLFDSAYGA “+++” 152 SLIRILQTI “+++” 153 SLLAELHVL “+++” 154 SLLAELHVLTV“++++” 155 SLMLEVPAL “+++” 156 SLNIGDVQL “+++” 157 SLNIRDFTM “+++” 158SLPEAPLDV “+++” 159 SLQEEKLIYV “+++” 160 SLSFLVPSL “+++” 161 SMDDGMINV“+++” 162 SMKDDLENV “++” 163 SQLDISEPYKV “+++” 164 SVHKGFAFV “+++” 165TLDDDLDTV “++” 166 TLDPNQVSL “++” 167 TLDTSKLYV “+++” 168 YLLDQSFVM“++++” 169 TLLLGLTEV “+++” 170 TLTFRVETV “+++” 171 TLVPPAALISI “+++” 172TLYDMLASI “+++” 173 TVIENIHTI “++” 174 VLAELPIIVV “+++” 175 FTVPRVVAV“+++” 176 VLAEQNIIPSA “+++” 177 VLDDRELLL “+++” 178 VLFFNVQEV “+++” 179VLLGLEMTL “+++” 180 LLKDGPEIGL “++” 181 VLLSIPFVSV “+++” 182 VLLSVPGPPV“+++” 183 VLMPTVYQQGV “+++” 184 VLSHNLYTV “+++” 185 VMDDQRDLI “++” 186VMDPTKILI “++++” 187 VMDTHLVNI “++++” 188 VMGDIPAAV “++” 189 VMLEMTPEL“+++” 190 VVMGTVPRL “+++” 191 YIFDGSDGGV “+++” 192 YIQEYLTLL “+++” 193YLDLSNNRL “+++” 194 YLDNVLAEL “++++” 195 YLGGFALSV “++++” 196 YLLLQTYVL“+” 197 YLQEVPILTL “++++” 198 YLTFLPAEV “+++” 199 YLVELSSLL “++++” 200YMFEEVPIVI “++++” 201 YQLELHGIEL “++++” 202 YVDDVFLRV “++++” 203ALLSSQLAL “+++” 204 GLLQINDKIAL “+++” 205 GLSQANFTL “+++” 206 HMQDVRVLL“+++” 207 IIADLDTTIMFA “+++” 208 ILLKTEGINL “+++” 209 ILQAELPSL “+++”210 KLLVQDFFL “+++” 211 LIDVKPLGV “+++” 214 RLQELTEKL “+++” 215VMQDIVYKL “+++” 216 WLAGDVPAA “+++” 217 ALDEPPYLTV “+++” 218 ALGEEWKGYVV“++” 219 ALLNLLESA “+++” 220 ALPEILFAKV “+++” 221 ALVSTIIMV “+” 222ALWELSLKI “+++” 223 ALWVSQPPEI “+++” 224 AMEALVVEV “+++” 225 ILQERELLPV“+++” 226 AMNISVPQV “+++” 227 FLAEASVMTQL “+++” 228 FLGGLSPGV “+++” 229FLLNLQNCHL “++++” 230 FLQDSKVIFV “+++” 231 FLYIRQLAI “+++” 232 FMHQIIDQV“++++” 233 GIIDINVRL “++” 234 GLDDAEYAL “++” 235 GLDDLLLFL “+++” 236GLLESGRHYL “+++” 237 GLQENLDVVV “++++” 238 GLVETELQL “+++” 239 ILAGEMLSV“+++” 240 ILARDILEI “+++” 241 ILGDILLKV “+++” 242 ILLGIQELL “+++” 243ILPTLEKELFL “+++” 244 ILQALAVHL “+++” 245 KIMDYSLLLGV “++++” 246KLDETGVAL “++” 247 KLKDRLPSI “++” 248 KTVEPPISQV “++” 249 LLPTGVFQV“+++” 250 LLVQEPDGLMV “+++” 251 LLYDNVPGA “+++” 252 NLLDPGSSYLL “+++”253 NLWSVDGEVTV “+++” 254 QLIPKLIFL “+++” 255 YLFEEAISM “+++” 256QLLPVSNVVSV “+++” 258 RLDYITAEI “+++” 259 RLLDEQFAVL “+++” 260SLDDVEGMSV “+++” 261 SLVEAQGWLV “+++” 262 SLWNAGTSV “+++” 263 SQWEDIHVV“+++” 264 TILDYINVV “+++” 265 TLLADDLEIKL “+++” 266 TLLDQLDTQL “++” 267TLLDWQDSL “+++” 268 TLLQVFHLL “+++” 269 TLTDEQFLV “++” 270 TVLPVPPLSV“+++” 271 VIRNIVEAA “++” 272 VLDELPPLI “+++” 273 VLGEYSYLL “+++” 274VLLEYHIAYL “+++” 275 VLLFIEHSV “+++” 276 VLNDGAPNV “+++” 277 VMILKLPFL“++” 278 YLDDLLPKL “+++” 279 YMAPEVVEA “+++” 280 YTLDSLYWSV “+++” 281NLLDDRGMTAL “+++” 282 LLRDGIELV “+++” 283 ILQPMDIHV “+++” 284 LLSAAEPVPA“++” 285 GVATAGCVNEV “++++” 286 FLLEDLSQKL “+++” 287 FLWEEKFNSL “++++”288 GLAESTGLLAV “+++” 289 ILEEQPMDMLL “+++” 291 ILLNEDDLVTI “+++” 292AAALIIHHV “+++” 293 ALDIMIPMV “+++” 294 ALLDQLHTLL “++++” 295 ALLQKLQQL“+++” 296 FIAPTGHSL “++” 297 FLVEPQEDTRL “+++” 298 IILPVEVEV “+++” 299ILEENIPVL “+++” 300 ILLNPAYDVYL “+++” 301 AASPIITLV “+++” 302 ILQDLTFVHL“+++” 303 ILSQPTPSL “+++” 304 LAIVPVNTL “+” 305 LLFPQIEGIKI “+++” 306VVAEELENV “++” 307 LLLTKPTEA “+++” 308 SLYDVSRMYV “++++” 309 ILYGTQFVL“+++” 310 LLSTLHLLV “+++” 311 LLVDVEPKV “+++” 312 LLYNSTDPTL “+++” 313LMADLEGLHL “+++” 314 LMKDCEAEV “+++” 315 LVYEAPETV “+++” 316 MLLEHGITL“+++” 317 NLLAHIWAL “+++” 318 NLQVTQPTV “+++” 319 QVIPQLQTV “+++” 320TIAPVTVAV “++++” 321 RLLEFELAQL “+++” 322 SLASIHVPL “+++” 323 SLDLFNCEV“++++” 324 SLYSALQQA “+++” 325 TLENGVPCV “+++” 326 VLAFLVHEL “++++” 327VLIKWFPEV “++++” 328 VLLPQETAEIHL “++++” 329 VLMDGSVKL “++++” 330VLMWEIYSL “+++” 331 VLWELAHLPTL “+++” 332 VMIQHVENL “+++” 333VTLEFPQLIRV “+++” 334 YLLEEKIASL “+++” 335 YLYQEQYFI “+++” 336 YMAVTTQEV“++++” 337 YMYEKESEL “++” 338 FLDMTNWNL “++++” 339 GLWGTVVNI “+++” 340KLLEEICNL “++++” 341 LLAELPASVHA “+++” 342 SLITPLQAV “+++” 344 VLAFENPQV“+++” 345 YLIEPDVELQRI “+++” 346 VLVQVSPSL “+++” 347 YLGPVSPSL “+++” 348ALAKPPVVSV “+++” 349 ALATHILSL “+++” 350 ALEDRVWEL “+++” 351 ALSEKLARL“+++” 352 ALVFELHYV “+++” 353 ATPMPTPSV “++” 354 FIMDDPAGNSYL “+++” 355FIWPMLIHI “+++” 356 FLHDHQAEL “++” 357 FLIQEIKTL “+++” 358 FLTDYLNDL“+++” 359 FMQDPMEVFV “+++” 360 HLIDTNKIQL “+++” 361 ILQEFESKL “+++” 362ILTELGGFEV “+++” 363 ITTEVVNELYV “+++” 364 KMDWIFHTI “+++” 365 LISPLLLPV“+++” 367 NLWSLVAKV “+++” 369 RLLDLENSLLGL “+++” 370 SIFASPESV “+++” 371SLADDSVLERL “+++” 372 SLFGPLPGPGPALV “+++” 373 TLLADQGEIRV “++” 374VLSVITEEL “+++” 375 VLWFKPVEL “+++” 376 VLYNQRVEEI “+++” 377 VVDGTCVAV“+++” 378 YILGKFFAL “+++” 379 YLAELVTPIL “+++” 380 YLDRKLLTL “+++” 381YLLEENKIKL “+++” 382 YLLPLLQRL “++++” 383 YLLREWVNL “++++” 384YMIGSEVGNYL “++++” 385 YTIPLAIKL “+++” Binding of HLA-class I restrictedpeptides to HLA-A*02:01 was ranged by peptide exchange yield: >10%= +; >20% = ++; >50 = +++; >75% = ++++

Example 6 Absolute Quantitation of Tumor Associated Peptides Presentedon the Cell Surface.

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and -specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.In addition to the isolation and relative quantitation of peptides asdescribed in EXAMPLE 1, the inventors did analyze absolute peptidecopies per cell as described in patent x. The quantitation of TUMAPcopies per cell in solid tumor samples requires the absolutequantitation of the isolated TUMAP, the efficiency of TUMAP isolation,and the cell count of the tissue sample analyzed. An overview on ourexperimental approach is described below.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labelledvariant of each peptide, i.e. two isotope-labelled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments. The calibration curves wereprepared in at least three different matrices, i.e. HLA peptide eluatesfrom natural samples similar to the routine MS samples, and eachpreparation was measured in duplicate MS runs. For evaluation, MSsignals were normalized to the signal of the internal standard and acalibration curve was calculated by logistic regression. For thequantitation of tumor-associated peptides from tissue samples, therespective samples were also spiked with the internal standard; the MSsignals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue In order todetermine the cell count of the tissue samples subjected to absolutepeptide quantitation, the inventors applied DNA content analysis. Thismethod is applicable to a wide range of samples of different origin and,most importantly, frozen samples (Alcoser et al., 2011; Forsey andChaudhuri, 2009; Silva et al., 2013). During the peptide isolationprotocol, a tissue sample is processed to a homogenous lysate, fromwhich a small lysate aliquot is taken. The aliquot is divided in threeparts, from which DNA is isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden,Germany). The total DNA content from each DNA isolation is quantifiedusing a fluorescence-based DNA quantitation assay (Qubit dsDNA HS AssayKit, Life Technologies, Darmstadt, Germany) in at least two replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of single healthy blood cells, with a range of defined cellnumbers, has been generated. The standard curve is used to calculate thetotal cell content from the total DNA content from each DNA isolation.The mean total cell count of the tissue sample used for peptideisolation is extrapolated considering the known volume of the lysatealiquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell number for selected peptides are shownin Table 12.

TABLE 12 Absolute copy numbers. The table lists the results of absolutepeptide quantitation in NSCLC tumor samples. Number of quantifiable SeqID Sequence Copy Number Category samples 62 GIIDGSPRL + 17 153 SLLAELHVL++ 17 The median number of copies per cell are indicated for eachpeptide: <100 = +; >=100 = ++; >=1,000 +++; >=10,000 = ++++. The numberof samples, in which evaluable, high quality MS data are available, isindicated.

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1. A method of eliciting an immune response in a patient who has cancer,comprising administering to the patient a composition comprising apopulation of activated T cells that selectively recognize cancer cellsthat present a peptide consisting of the amino acid sequence ofFVYGESVEL (SEQ ID NO: 10), wherein the activated T cells are produced bycontacting T cells with the peptide loaded human class I MHC moleculeexpressed on the surface of an antigen-presenting cell, wherein saidcancer is selected from the group consisting of chronic lymphocyticleukemia (CLL), acute myelogenous leukemia, bile duct cancer, braincancer, breast cancer, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, hepatocellular cancer, Merkel cellcarcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer,ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer,small cell lung cancer, urinary bladder cancer, and uterine cancer. 2.The method of claim 1, wherein the T cells are autologous to thepatient.
 3. The method of claim 1, wherein the T cells are obtained froma healthy donor.
 4. The method of claim 1, wherein the T cells areobtained from tumor infiltrating lymphocytes or peripheral bloodmononuclear cells.
 5. The method of claim 1, wherein the activated Tcells are expanded in vitro.
 6. The method of claim 1, wherein thepeptide is in a complex with the class I MHC molecule.
 7. The method ofclaim 1, wherein the composition further comprises an adjuvant.
 8. Themethod of claim 7, wherein the adjuvant is selected from the groupconsisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivates, poly-(I:C) andderivates, RNA, sildenafil, particulate formulations with poly(lactidco-glycolid) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7,IL-12, IL-13, IL-15, IL-21, and IL-23.
 9. The method of claim 1, whereinthe antigen presenting cell is infected with a recombinant virusexpressing the peptide.
 10. The method of claim 9, wherein the antigenpresenting cell is a dendritic cell or a macrophage.
 11. The method ofclaim 5, wherein the expansion is in the presence of an anti-CD28antibody and IL-12.
 12. The method of claim 1, wherein the population ofactivated T cells comprises CD8-positive cells.
 13. The method of claim1, wherein the contacting is in vitro.
 14. The method of claim 1,wherein the immune response comprises a cytotoxic T cell response. 15.The method of claim 1, wherein the immune response is capable of killingcancer cells that present a peptide consisting of the amino acidsequence of FVYGESVEL (SEQ ID NO: 10).
 16. A method of killing cancercells, comprising eliciting the immune response of claim
 1. 17. Themethod of claim 1, wherein the cancer is non-Hodgkin lymphoma.
 18. Amethod of treating a patient who has cancer, comprising administering tothe patient a composition comprising a population of activated T cellsthat selectively recognize cancer cells that present a peptideconsisting of the amino acid sequence of FVYGESVEL (SEQ ID NO: 10),wherein the activated T cells are produced by contacting T cells withthe peptide loaded human class I MHC molecule expressed on the surfaceof an antigen-presenting cell, wherein said cancer is selected from thegroup consisting of chronic lymphocytic leukemia (CLL), acutemyelogenous leukemia, bile duct cancer, brain cancer, breast cancer,colorectal carcinoma, esophageal cancer, gallbladder cancer, gastriccancer, hepatocellular cancer, Merkel cell carcinoma, melanoma,non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, renal cell cancer, small cell lungcancer, urinary bladder cancer, and uterine cancer.
 19. The method ofclaim 18, wherein the population of activated T cells comprisesCD8-positive cells.
 20. The method of claim 18, wherein the cancer isnon-Hodgkin lymphoma.